Electron-deficient nitrogen heterocycle-substituted fluorescein dyes

ABSTRACT

The invention provides compositions electron-deficient nitrogen heterocycle-substituted fluorescein dyes and methods in which the dyes are conjugated to substrates and used as detection labels in molecular biology experiments. The electron-deficient nitrogen heterocycles include pyridine, quinoline, pyrazine, and the like. Substrates include polynucleotides, nucleosides, nucleotides, peptides, proteins, carbohydrates, and ligands.

FIELD OF THE INVENTION

The invention relates generally to the field of fluorescent dyecompounds useful as labelling reagents to prepare molecular probes. Morespecifically, this invention relates to fluorescein dyes with a xanthenering structure and electron deficient nitrogen heterocycle substituents.

BACKGROUND

The non-radioactive detection of biological analytes utilizingfluorescent labels is an important technology in modern analyticalbiotechnology. By eliminating the need for radioactive labels, safety isenhanced and the environmental impact and costs associated with reagentdisposal are greatly reduced. Examples of methods utilizing suchfluorescent detection methods include automated DNA sequencing,oligonucleotide probe methods, detection of polymerase-chain-reactionproducts, immunoassays, and the like.

In many important applications, it is advantageous to employ multiplespectrally distinguishable fluorescent labels in order to achieveindependent detection of a plurality of spatially overlapping analytes,i.e. multiplex fluorescent detection. Examples of methods utilizingmultiplex fluorescent detection include single-tube multiplex DNA probeassays, PCR, single nucleotide polymorphisms, immunoassays, andmulti-color automated DNA sequencing. The number of reaction vessels maybe reduced thereby simplifying experimental protocols and facilitatingthe production of application-specific reagent kits. In the case ofmulti-color automated DNA sequencing, multiplex fluorescent detectionallows for the analysis of multiple nucleotides in a singleelectrophoresis lane thereby increasing throughput over single-colormethods and reducing uncertainties associated with inter-laneelectrophoretic mobility variations. Automated four-color Sanger-typeDNA sequencing has enabled entire genome characterization at themolecular level.

Assembling a set of multiple spectrally distinguishable fluorescentlabels useful for multiplex fluorescent detection is problematic.Multiplex fluorescent detection imposes at least six severe constraintson the selection of component fluorescent dye labels, particularly forapplications requiring a single excitation light source, anelectrophoretic separation, and/or treatment with enzymes, e.g.,automated DNA sequencing. First, it is difficult to find a set ofstructurally similar dyes whose emission spectra are spectrallyresolved, since the typical emission band half-width for organicfluorescent dyes is about 40-80 nanometers (nm). Second, even if dyeswith non-overlapping emission spectra are identified, the set may stillnot be suitable if the respective fluorescent quantum efficiencies aretoo low. Third, when several fluorescent dyes are used concurrently,simultaneous excitation becomes difficult because the absorption bandsof the dyes are usually widely separated. Fourth, the charge, molecularsize, and conformation of the dyes must not adversely affect theelectrophoretic mobility of the analyte. Fifth, the fluorescent dyesmust be compatible with the chemistry used to create or manipulate theanalyte, e.g., DNA synthesis solvents and reagents, buffers, polymeraseenzymes, ligase enzymes, and the like. Sixth, the dye must havesufficient photostability to withstand laser excitation.

Currently available multiple dye sets suitable for use in four-colorautomated DNA sequencing applications require blue or blue-green laserlight to adequately excite fluorescence emissions from all of the dyesmaking up the set, e.g., argon-ion lasers. As lower cost red lasersbecome available, a need develops for fluorescent dye compounds andtheir conjugates which satisfy the above constraints and are excitableby laser light having a wavelength above about 500 nm.

SUMMARY

The present invention relates to dye compounds suitable for the creationof sets of spectrally-resolvable fluorescent labels useful formulti-color fluorescent detection.

Generally the dyes of the invention comprise a fluorescein-type,xanthene ring structure I:

-   -   substituted with at least one electron-deficient nitrogen        heterocycle linked to the fluorescein ring system at R¹, R², R³,        R⁴, R⁵, or R⁷.

R¹, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R⁷ isbenzo or heterocycle.

R², when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle.

R³, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle.

R⁴, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R⁵ isbenzo or heterocycle.

R⁵, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R⁴ isbenzo or heterocycle.

R⁷, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R⁴ isbenzo or heterocycle.

R⁶ can be (C₁-C₆) alkyl, (C₂-C₆) alkene, (C₂-C₆) alkyne, cyano,heterocyclic aromatic, phenyl, and substituted phenyl having thestructure II:

-   -   wherein X¹, X², X³, X⁴ and X⁵ taken separately are H, Cl, F,        (C₁-C₆) alkyl, (C₂-C₆) alkene, (C₂-C₆) alkyne, CO₂H, SO₃H,        CH₂OH, or reactive linking group.

In one aspect, the invention provides a method of labelling a substratewith a fluorescein dye of structure I, where the substrate reacts withthe reactive linking group of the dye and a substrate-dye conjugate isformed. Substrate dye-labelled conjugates comprise the electrondeficient nitrogen heterocycle-substituted fluorescein dye, according toI, and a substrate, i.e. another molecule or substance. The substratemay be labelled with one or more dyes of the invention, which may be thesame or different. Fluorescence from the dyes provides detectablesignals across a spectral range, enabling differentiation of differentlylabelled substrates in a single sample or mixture.

In one embodiment, the electron deficient nitrogenheterocycle-substituted fluorescein dye is covalently conjugated toanother dye compound to form an energy-transfer dye compound.

In another embodiment, the electron deficient nitrogenheterocycle-substituted fluorescein dye is covalently conjugated to anucleoside, nucleotide, nucleoside and nucleotide analog, polynucleotideand polynucleotide analog to form labelled conjugates therewith.

In yet another aspect, the invention provides phosphoramidite reagentsincluding the electron deficient nitrogen heterocycle-substitutedfluorescein dyes of the invention.

In another aspect, the invention provides various methods forsynthesizing oligonucleotides labelled with electron-deficient nitrogenheterocycle-substituted fluorescein dyes, and employing the dyes fordetection of fluorescent labelled polynucleotides.

In another aspect, the invention provides kits comprisingelectron-deficient nitrogen heterocycle-substituted fluorescein dyes andreagents useful for labelling molecules and/or for performing assayssuch as DNA sequencing and amplification, e.g. polymerase chainreaction.

The electron-deficient nitrogen heterocycle-substituted fluorescein dyesof the invention provide significant advantages over currently knownfluorescein dyes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structures of the compounds 1-5

FIG. 2 shows the structures of the compounds 6-12

FIG. 3 shows the structures of the compounds 13 and 14

FIG. 4 shows the structures of the compounds 15 and 16

FIG. 5 shows the structures of the compounds 17-22

FIG. 6 shows the structures of the compounds 23-25

FIG. 7 shows the structures of the compounds 26-28

FIG. 8 shows the structures of the compounds 29-33

FIG. 9 shows the structures of the compounds 34-36

FIG. 10 shows the structures of the compounds 37-41

FIG. 11 shows a representative collection of electron-deficient nitrogenheterocycles

FIG. 12 shows fluorimetry scans of compound 19; Excitation max. 530 nm,Emission max. 554 nm. in 1×TBE at room temperature

FIG. 13 shows fluorimetry scans of compound 22; Excitation max. 531.5nm, Emission max. 556 nm. in 1×TBE at room temperature

FIG. 14 shows a fluorimetry scan of compound 25; Emission max. 562.5 nm.in 1×TBE at room temperature

FIG. 15 shows fluorimetry scans of compound 33; Excitation max. 545.5nm, Emission max. 571.5 nm. in 1×TBE at room temperature

FIG. 16 shows fluorescent detection of labelled sequencing fragmentsfrom the ABI PRISM 310. Base 183 to base 276 region from G terminationsequence of pGEM target using −21M13 primer. Reagents: POP6electrophoresis medium, Taq FS polymerase, dNTP mix 25 pmole each dATP,dCTP, dITP, dTTP. Top panel: dNTP mix and terminator 3′FddGTP-EO-13.Bottom panel: dNTP mix and energy-transfer dye terminator3′FddGTP-EO-6FAM-Bn-dR110.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Reference will now be made in detail to the preferred embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. While the invention will be described in conjunction with thepreferred embodiments, it will be understood that they are not intendedto limit the invention to those embodiments. On the contrary, theinvention is intended to cover all alternatives, modifications, andequivalents, which may be included within the scope of the presentinvention as defined by the appended claims.

Definitions

Unless stated otherwise, the following terms and phrases as used hereinare intended to have the following meanings:

“Nucleobase” means a nitrogen-containing heterocyclic moiety capable offorming Watson-Crick hydrogen bonds with a complementary nucleobase ornucleobase analog, e.g. a purine, a 7-deazapurine, or a pyrimidine.Typical nucleobases are the naturally occurring nucleobases adenine,guanine, cytosine, uracil, thymine, and analogs of the naturallyoccurring nucleobases, e.g. 7-deazaadenine, 7-deazaguanine, inosine,nebularine, nitropyrrole, nitroindole, 2-amino-purine,2,6-diamino-purine, hypoxanthine, pseudouridine, pseudocytidine,pseudoisocytidine, 5-propynyl-cytidine, isocytidine, isoguanine,7-deaza-quanine, 2-thio-pyrimidine, 6-thio-guanine, 4-thio-thymine,4-thio-uracil, O⁶-methyl-guanine, N⁶-methyl-adenine, O⁴-methyl-thymine,5,6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, andethenoadenine (Fasman, Practical Handbook of Biochemistry and MolecularBiology, pp. 385-394, CRC Press, Boca Raton, Fla. (1989)).

“Nucleoside” means a compound comprising a nucleobase linked to a C-1′carbon of a ribose sugar. The ribose may be substituted orunsubstituted. Substituted ribose sugars include, but are not limitedto, those riboses in which one or more of the carbon atoms, preferablythe 3′-carbon atom, is substituted with one or more of the same ordifferent —R, —OR, —NRR or halogen groups, where each R is independently—H, (C₁-C₆) alkyl or (C₅-C₁₄) aryl. Particularly preferred riboses areribose, 2′-deoxyribose, 2′,3′-dideoxyribose, 3′-haloribose,3′-fluororibose, 3′-chlororibose, and 3′-alkylribose. When thenucleobase is A or G, the ribose sugar is attached to the N⁹-position ofthe nucleobase. When the nucleobase is C, T or U, the pentose sugar isattached to the N¹-position of the nucleobase (Kornberg and Baker, DNAReplication, 2^(nd) Ed., Freeman, San Francisco, Calif., (1992)).

“Nucleotide” means a phosphate ester of a nucleoside, as a monomer unitor within a polynucleotide. Nucleotides are sometimes denoted as “NTP”,or “dNTP” and “ddNTP” to particularly point out the structural featuresof the ribose sugar. “Nucleotide 5′-triphosphate” refers to a nucleotidewith a triphosphate ester group at the 5′ position. The triphosphateester group may include sulfur substitutions for the various oxygens,e.g. α-thio-nucleotide 5′-triphosphates.

As used herein, the terms “polynucleotide” or “oligonucleotide”encompass any polymer compound comprised of nucleosides, nucleotides oranalogs thereof. “Oligonucleotide” and “polynucleotide”, usedinterchangeably, mean single-stranded and double-stranded polymers ofnucleotide monomers, including 2′-deoxyribonucleotides (DNA) andribonucleotides (RNA). An oligonucleotide may be composed entirely ofdeoxyribonucleotides, entirely of ribonucleotides, or chimeric mixturesthereof, linked by internucleotide phosphodiester bond linkages, orinternucleotide analogs, and associated counterions, e.g., H⁺, N₄ ⁺,trialkylammonium, Mg²⁺, Na⁺ and the like. Oligonucleotides may becomprised of nucleobase and sugar analogs. Polynucleotides typicallyrange in size from a few monomeric units, e.g. 5-40 when they arecommonly referred to as oligonucleotides, to several thousands ofmonomeric nucleotide units. Unless denoted otherwise, whenever anoligonucleotide sequence is represented, it will be understood that thenucleotides are in 5′ to 3′ order from left to right and that “A”denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, unless otherwise noted.

The term “Watson/Crick base-pairing” refers to the hydrogen-bonding basepairing commonly observed in double-stranded DNA.

“Attachment site” refers to a site on a moiety, e.g. a fluorescein dye,a nucleotide, or an oligonucleotide, to which a linker is covalentlyattached.

“Linker” refers to a moiety that links a dye to a substrate, e.g. anoligonucleotide, or one dye to another, e.g. in an energy-transfer dyepair.

“Alkyl” refers to a saturated or unsaturated, straight-chain, branched,or cyclic hydrocarbon radical derived by the removal of one hydrogenatom from a single carbon atom of a parent alkane, alkene, or alkyne.Typical alkyl groups include, but are not limited to, methyl, ethyl,propyl, butyl, and the like. Typical alkyl groups include, but are notlimited to, methyl (—CH₃); ethyls such as ethanyl (—CH₂—CH₃), ethenyl(—CH═CH₂), ethynyl (—C≡CH); propyls such as propan-1-yl (—CH₂—CH₂—CH₃),propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl (—CH═CH—CH₂),prop-1-en-2-yl, prop-2-en-1-yl (—CH₂—CH═CH₂), prop-2-en-2-yl,cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl (—C≡C—CH₃),prop-2-yn-1 -yl (—CH₂—C≡CH), etc.; butyls such as butan-1-yl(—CH₂—CH₂—CH₂—CH₃), butan-2-yl, cyclobutan-1-yl, but-1-en-1-yl(—CH═CH₂—CH₂—CH₃), but-1-en-2-yl, but-2-en-1-yl (—CH₂—CH═CH₂—CH₃),but-2-en-2-yl, buta-1,3-dien-1-yl (—CH═CH—CH═CH₂), buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl (—C≡C—CH₂—CH₃), but-1-yn-3-yl, but-3-yl-1-yl(—CH₂—CH₂—C≡CH), etc.; and the like. In preferred embodiments, the alkylgroups are (C₁-C₆) alkyl, with (C₁-C₃) being particularly preferred.

“Alkoxy” means —OR where R is (C₁-C₆) alkyl.

“Alkyldiyl” refers to a saturated or unsaturated, branched, straightchain or cyclic hydrocarbon radical of 1-12 carbon atoms and having twomonovalent radical centers derived by the removal of two hydrogen atomsfrom the same or two different carbon atoms of a parent alkane, alkeneor alkyne. Typical alkyldiyl radicals include, but are not limited to,methano (—CH₂—); 1,2-ethyldiyl; 1,3-propyldiyl; 1,4-butyldiyl; and thelike.

“Aryl” refers to a monovalent aromatic hydrocarbon radical of 6-20carbon atoms derived by the removal of one hydrogen atom from a singlecarbon atom of a parent aromatic ring system. Typical aryl groupsinclude, but are not limited to, radicals derived from benzene,substituted benzene, naphthalene, anthracene, biphenyl, and the like.

“Aryldiyl” refers to an unsaturated cyclic or polycyclic hydrocarbonradical of 6-20 carbon atoms having a conjugated resonance electronsystem and at least two monovalent radical centers derived by theremoval of two hydrogen atoms from two different carbon atoms of aparent aryl compound.

“Substituted alkyl”, “substituted alkyldiyl”, “substituted aryl” and“substituted aryldiyl” refer to alkyl, alkyldiyl radicals, aryl andaryldiyl respectively, in which one or more hydrogen atoms are eachindependently replaced with another substituent. Typical substituentsinclude, but are not limited to, —X, —R, —O⁻, —OR, —SR, —S^(—), —NRR,═NR, —CX₃, —CN, —OCN, —SCN, —NCO, —NCS, —NO, —NO₂, ═N₂, —N₃, —S(O)₂O⁻,—S(O)₂OH, —S(O)₂R, —P(O)(O⁻)₂, —P(O)(OH)₂, —C(O)R, —C(O) X, —C(S)R,—C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR, —C(S)NRR and—C(NR)NRR, where each X is independently a halogen and each R isindependently —H, alkyl, aryl, or heterocycle. Particularly preferredsubstituents are halogen, —OS(O)₂OR, —S(O)₂ OR, —S(O)₂R, —S(O)₂NR,—S(O)R, —OP(O)O₂RR, —P(O)O₂RR, —C(O)OR, —NRR, —NRRR, —NC(O)R, —C(O)R,—C(O)NRR, —CN, —O and —OR, wherein R is independently selected from thegroup consisting of —H, alkyl, aryl, heteroaryl, heterocycle and linkinggroup.

“Reactive linking group” refers to a chemically reactive moiety, e.g. anucleophile or electrophile, capable of reacting with another moleculeto form a covalent bond, or linkage.

“Heterocycle” refers to a molecule with a ring system in which one ormore ring atoms have been replaced with a heteroatom, e.g. nitrogen,oxygen, and sulfur.

“Electron-deficient nitrogen heterocycle substituent” refers to amonovalent electron-deficient nitrogen heterocycle derived by theremoval of one hydrogen atom from a single atom of the ring system tojoin the heterocycle to the fluorescein dyes of the invention (Joule,etal, Heterocyclic Chemistry, 3rd Ed., Stanley Thornes Publisher, Ltd.,Cheltenham, U.K. (1998); Acheson, R., An Introduction to the Chemistryof Heterocyclic Compounds, 2nd Ed. Interscience Publishers, division ofJohn Wiley & Sons, New York (1967)). Several examplaryelectron-deficient nitrogen heterocycles are set forth in FIG. 11.

“Substrate” is an entity to which dye compounds of the present inventionare attached. Substrates include, but are not limited to a (i)polynucleotide, (ii) nucleoside and nucleotide, (iii) peptide andprotein, (iv) carbohydrate, (v) ligand, and (vi) any analog of thepreceding (i) to (v).

“Enzymatically incorporatable” refers to a property of a nucleotide inwhich it is capable of being enzymatically incorporated onto theterminus, e.g. 3′, of a nascent polynucleotide chain through the actionof a polymerase enzyme.

“Terminator” means an enzymatically incorporatable nucleotide whichprevents subsequent incorporations of nucleotides to the resultingpolynucleotide chain and thereby halt polymerase extension. Typicalterminators lack a 3′-hydroxyl substituent and include2′,3′-dideoxyribose, 2′,3′-didehydroribose, and 2′,3′-dideoxy,3′-haloribose, e.g. 3′-fluoro. Alternatively, a ribofuranose analogcould be used, such as arabinose. Exemplary nucleotide terminatorsinclude 2′,3′-dideoxy-β-D-ribofuranosyl, β-D-arabinofuranosyl,3′-deoxy-β-D-arabinofuranosyl, 3′-amino-2′,3′-dideoxy-β-D-ribofuranosyl,and 2′,3′-dideoxy-3′-fluoro-β-D-ribofuranosyl (Chidgeavadze etal. (1984)Nucleic Acids Res., 12: 1671-1686; and Chidgeavadze etal. (1985) FEB.Lett., 183: 275-278). Nucleotide terminators also include reversiblenucleotide terminators (Metzker etal. (1994) Nucleic Acids Res., 22(20):4259).

“Enzymatically extendable” means a property of a nucleotide in which itis enzymatically incorporatable at the terminus of a polynucleotide andthe resulting extended polynucleotide can undergo subsequentincorporations of nucleotides or nucleotide analogs.

“Internucleotide analog” means a phosphate ester analog ofoligonucleotides which include, but are not limited to,alkylphosphonates, methylphosphonates, phosphoramidates,phosphotriesters, phosphorothioates, phosphorodithioates,phosphoroamidates. Internucleotide analogs also include non-phosphateanalogs where the sugar/phosphate group is replaced by amide linkages,such as 2-aminoethylglycine units, referred to as PNA (Nielsen, etal,(1991) “Sequence-selective recognition of DNA by strand displacementwith a thymidine-substituted polyamide”, Science 254:1497-1500).

“Target sequence” means a polynucleotide, DNA or RNA, single-stranded ordouble-stranded that is the subject of hybridization with a primer orprobe, enzymatic activity, or detection.

“Spectrally Resolvable” means, in reference to a set of fluorescentdyes, that the fluorescence emission bands of the respective dyes aresufficiently distinct, i.e., sufficiently non-overlapping, that thedyes, either alone or when conjugated to other compounds (labelled), aredistinguishable from one another on the basis of their fluorescencesignals using standard photodetection systems such as photodetectorsemploying a series of band pass filters and photomultiplier tubes,charged-coupled devices (CCD), spectrographs, etc. (Wheeless et al.,(1985) Flow Cytometry: Instrumentation and Data Analysis, pp. 21-76,Academic Press, New York). Preferably, all of the dyes comprising aspectrally resolvable set of dyes are excitable by a single lightsource.

“Mobility-Matched” refers to a set of fluorescent dyes that, when usedto label polynucleotides of equal lengths, yields differentiallylabelled polynucleotides having substantially similar electrophoreticmobilities. Typically, the relative electrophoretic mobilities ofpolynucleotides labelled with a set of mobility-matched dyes will varyby less than about one-half nucleotide. Preferably, the mobility-matcheddyes are spectrally resolvable, as previously defined.

“Relative photostability” means the chemical stability of fluorescentdyes relative to a standard reference, 5-carboxyfluorescein, as measuredby exposure to high-intensity white light with sampling measurement ofremaining dye at its absorption maxima. Equal optical density units ofthe test dye and the reference are subjected in parallel to light, as acorrelative test for stability under the laser-induced fluorescencecommon to automated DNA sequencing and fragment analysis applications.

Dyes

In a first aspect, the present invention comprises a novel class offluorescein-type, xanthene ring compounds, according to structure I:

-   -   substituted with at least one electron-deficient nitrogen        heterocycle linked to the fluorescein ring system at R¹, R², R³,        R⁴, R⁵, or R⁷. The electron-deficient nitrogen heterocycle may        be linked to the fluorescein ring system through a carbon-carbon        bond or a carbon-nitrogen bond. All molecular structures        provided throughout this disclosure are intended to encompass        not only the exact electronic structure presented, but also        include all resonant structures, tautomers, enantiomers,        diastereomers, and protonation states thereof

R¹, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R⁷ isbenzo or heterocycle.

R², when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle.

R³, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle.

R⁴, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R⁵ isbenzo or heterocycle.

R⁵, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R⁴ isbenzo or heterocycle.

R⁷, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substitutedalkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle, or when taken together with R′ isbenzo or heterocycle.

R⁶ is selected from the group consisting of (C₁-C₆) alkyl, (C₂-C₆)alkene, (C₂-C₆) alkyne, cyano, heterocyclic aromatic, phenyl, andsubstituted phenyl having the structure II:

-   -   wherein X¹, X², X³, X⁴ and X⁵ taken separately are H, Cl, F,        (C₁-C₆) alkyl, (C₂-C₆) alkene, (C₂-C₆) alkyne, CO₂H, SO₃H,        CH₂OH, or reactive linking group.

Particularly preferred dyes according to I include where R⁴ takentogether with R⁵ forms a fused ring, e.g. benzo. Likewise preferred iswhere R¹ taken together with R⁷ forms a fused ring, e.g. benzo.

Preferred dyes include where R¹, R², R³ and R⁴, each taken separately,are phenyl and substituted phenyl, naphthyl, substituted naphthyl,fluoro, chloro, 2-pyridyl, 3-pyridyl, 2-quinolyl or 3-quinolyl. R⁵ andR⁷ are preferably hydrogen.

Another class of particularly preferred dyes according to II includesthose where the substituted phenyl has structure IIa:

A preferred embodiment of IIa is where one of X³ and X⁴ is a reactivelinking group and the other is hydrogen. A particularly preferredembodiment of IIa is where one of X³ and X⁴ is carboxyl and the other ishydrogen.

Various aspects of the above-described invention enable one or more ofthe following important advantages over known fluorescent dye compoundsuseful for multiplex fluorescent detection: (1) the emission spectra ofthe subject dye compounds can be modulated by minor variations in thetype and location of the electron-deficient nitrogen heterocycle and/oraryl-substituents, allowing for the creation of dye sets having similarabsorption characteristics yet spectrally resolvable fluorescenceemission spectra; (2) the subject dye compounds may be easily attachedto substrates without compromising their favorable fluorescenceproperties; (3) the subject dye compounds have narrow emissionbandwidths, i.e., the emission bandwidth has a full-width at half themaximum (FWHM) emission intensity of below about 45 nm; (4) the subjectdye compounds are highly soluble in buffered aqueous solution whileretaining a high quantum yield; (5) the subject dye compounds arerelatively photostable; and (6) the subject dye compounds haverelatively large extinction coefficients, i.e., greater than about50,000 (Benson etal “Aromatic-substituted xanthene dyes”, U.S. Pat. No.6,008,379, issued Dec. 28, 1999). Narrow emission bandwidths, asexemplified by FWHM, are desirable as facilitating spectral resolutionin a mixture of substrates labelled with more than one (a set)fluorescent dye. Photostability is an important property in thatsubstrates, e.g. polynucleotides, labelled with the dyes of theinvention may be subjected to intense laser light to induce fluorescencefor detection. Photobleaching, or other chemical degradation mechanisms,diminish or prevent detection of substrates labelled with dyes of lessthan optimal photostability.

FIG. 11 shows a representative sample of electron-deficient nitrogenheterocycles which may be used as substituents on the fluorescein dyesof the invention. These heterocycle substituents have direct effects onimportant spectral properties of fluorescent dyes. These effects areexemplified by the spectral properties of fluorescein dyes of thepresent invention such as: (i) excitation, emission, and-absorptionmaxima and spectra, (ii) photostability, (iii) quantum efficiency, and(iv) energy-transfer efficiency.

Electron-deficient nitrogen heterocycles may have one 5- or 6-memberedring bearing one or more nitrogen atoms in the ring (FIG. 11). The 5- or6-membered ring may be fused to a second aromatic ring, e.g. a benzo orsubstituted-benzo ring, or saturated ring, e.g. cyclopentyl orcyclohexyl. The heterocycle may bear other heteroatoms, e.g. oxygen orsulfur, in the ring system.

Preferred electron-deficient nitrogen heterocycles include, but are notlimited to those in FIG. 11 and 2-pyridyl, 3-pyridyl, 4-pyridyl,2-quinolyl, 3-quinolyl, 4-quinolyl, 2-imidazole, 4-imidazole,3-pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline,pthalazine, quinazoline, quinoxaline, 3-(1,2,4-N)-triazolyl,5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O, 3N)-oxazole, 5-(1-O,3-N)-oxazole, 4-(1-S, 3-N)-thiazole, 5-(1-S, 3-N)-thiazole,2-benzoxazole, 2benzothiazole, benzotriazole, 4-(1,2,3-N)-benzotriazine,and benzimidazole. The heterocycle may be attached to the xanthene ringsystem directly by a bond with a carbon atom of the heterocycle ring.Alternatively, the heterocycle may be attached to the xanthene ringsystem through an unsaturated linker which extends delocalization ofaromaticity between the heterocycle and the xanthene ring system.Linkers that permit extensive electronic delocalization include, but arenot limited to: (i) olefinic; —CR═CR—, (ii) polyolefinic; —(CR═CR)_(n)—where n is 2 to 10, (iii) acetylenic; —C≡C—, (iv) polyacetylenic;—(C≡C)_(n)— where n is 2 to 10, (v) squarine, and (vi) other cyclicconjugated linkers. R is selected from the group consisting of hydrogen,(C₁-C₆) alkyl, halogen, fluorine, chlorine, CN, CF₃, aryl, andsubstituted aryl. The geometry of the olefinic linkages may be cis ortrans, E or Z.

Energy Transfer Dyes

In another aspect, the present invention comprises energy transfer dyecompounds incorporating the electron-deficient nitrogenheterocycle-substituted fluorescein dye compounds of structure I.Generally, the energy transfer dyes of the present invention include adonor dye which absorbs light at a first wavelength and emits excitationenergy in response, an acceptor dye which is capable of absorbing theexcitation energy emitted by the donor dye and fluorescing at a secondwavelength in response, and a linker which attaches the donor dye to theacceptor dye, the linker being effective to facilitate efficient energytransfer between the donor and acceptor dyes and to maintain a highemission quantum yield of the acceptor dye (Lee, etal “Energy transferdyes with enhanced fluorescence”, U.S. Pat. No. 5,800,996, issued Sep.1, 1998; Lee, etal “Energy transfer dyes with enhanced fluorescence”,U.S. Pat. No. 5,945,526, issued Aug. 31, 1999; Mathies, etal“Fluorescent labels and their use in separations”, U.S. Pat. No.5,654,419, issued Aug. 5, 1997). In the energy transfer dye of theinvention, at least one of the donor acceptor dyes is anelectron-deficient nitrogen heterocycle-substituted fluorescein dye.

Energy transfer dyes have advantages for use in the simultaneousdetection of multiple labelled substrates in a mixture, such as DNAsequencing. A single donor dye can be used in a set of energy transferdyes so that each dye pair has strong absorption at a common wavelength.By then varying the acceptor dye in the energy transfer set, theacceptor dyes can be spectrally resolved by their respective emissionmaxima. Energy transfer dyes also provide a larger effective Stokesshift than non-energy transfer dyes. The Stokes shift is the differencebetween the excitation maximum, the wavelength at which the donor dyemaximally absorbs light, and the emission maximum, the wavelength atwhich the acceptor maximally emits light.

In a preferred embodiment, the linker between the donor dye and acceptordye includes a functional group which gives the linker some degree ofstructural rigidity, such as an alkene, diene, an alkyne, a five and sixmembered ring having at least one unsaturated bond or a fused ringstructure. The donor dye and the acceptor dye of the energy transfer dyemay be attached by linkers which have the structures:

wherein n is 1 or 2.

The attachment sites of the linker between the donor dye and acceptordye of an energy transfer dye may be at any position R¹-R⁷ where one orboth of the donor dye and acceptor dye is a dye of the presentinvention. Preferred attachment sites include R², R³, X³, and X⁴.

The energy transfer dye compound is covalently attached to a substratethrough a linker. The linker may be alkyldiyl (C₁-C₁₂) or aryldiyl(C₆-C₂₀) and bearing functional groups including amide, carbamate, urea,thiourea, phosphate, phosphorothioate, and the like. Preferred linkersinclude 1,2-ethyldiyl and 1,6-hexyldiyl. The attachment sites of thelinker between the energy transfer dye and the substrate may be at anyposition R¹-R⁷ on the energy transfer dye, where one or both of thedonor dye and acceptor dye is a dye of the present invention. Preferredattachment sites include R², R³, X³, and X⁴. Where the substrate is anucleoside or nucleotide, a preferred attachment site is on thenucleobase. Where the substrate is an oligonucleotide, preferredattachment sites include the 3′ and 5′ terminii. Where the substrate isa peptide or protein, preferred attachment sites include the amino andcarboxyl terminii.

Methods of Synthesis

Several synthetic methods are available for the synthesis of thefluorescein dyes of the invention. A preferred method of synthesis ofintermediates is via the palladium-catalyzed Suzuki boronate couplingreaction whereby an aryl boronic acid is coupled with an aryl halide togive a biaryl product with regioselectivity (Zhang, etal (1998) “Baseand cation effects on the Suzuki cross-coupling of bulky arylboronicacid with halopyridines: synthesis of pyridylphenols”, J. Org. Chem.63:6886-90; Martin, etal (1993) “Palladium-catalyzed cross-couplingreactions of organoboronic acids with organic electrophiles”, ActaChemica Scan. 47:221-30; Aliprantis, etal (1994) “Observation ofcatalytic intermediates in the Suzuki reaction by electrospray massspectrometry”, J. Am. Chem. Soc. 116:6985-86; Thompson, etal (1984) “Ageneral synthesis of 5-arylnicotinates”, J. Org. Chem. 49:5237-43).

Cyclization substrate 5 is synthesized in FIG. 1. The Suzuki reaction isiterated, first by coupling 1,3-dimethoxyphen-2-yl boronic acid with2-bromotoluene with tetrakis(triphenylphosphine) palladium catalysis togive biphenyl compound 1. Regioselective bromination of 1 gave 2,followed by Suzuki reaction with pyridine-3-boronic acid under palladiumcatalysis to yield 3. Demethylation of 3 with hydrobromic acid andacetic acid gave 4, followed by Friedel-Crafts acylation with2,5-dichlorotrimellitic anhydride to yield 5.

Brominated naphthyl compound 8 serves as a common intermediate for thesynthesis of cyclization intermediates 11 and 12, illustrated in FIG. 2.Suzuki coupling of pyridine-3-boronic acid and tol-2-yl boronic acidgave 9 and 10, respectively, which were demethylated to 11 and 12.

Cyclization of an equimolar mixture of intermediates 5 and 11 inmethanesulfonic acid at 100° C. gave 30% yield of dye Z13 (FIG. 3) afterchromatography (Example 13). Likewise, 14 was formed by cyclization of 5with 12 (Example 14). Dye 15 (FIG. 4) was formed by cyclization of 5 and2-fluoro-1,3-dihydroxynaphthalene (Benson, etal “Asymmetricbenzoxanthene dyes”, U.S. Pat. No. 5,840,999, issued Nov. 24, 1998).Symmetric dye 16 (FIG. 4) was synthesized by double cyclization of twomolar equivalents of 4 and one molar equivalent 2,5-dichlorotrimelliticanhydride (Example 16).

By the same methods, electron-deficient nitrogen heterocycle-substitutedfluorescein dyes 19, 22 (FIG. 5), 25 (FIG. 6), 28 (FIG. 7), 33 (FIG. 8),36 (FIG. 9, and 41 (FIG. 10) were synthesized (Examples 17-41).

Labelling Reagents of the Dyes

The present invention comprises labelling reagents whereinelectron-deficient nitrogen heterocycle-substituted fluorescein dyes arein reactive form to react with substrates. In another aspect, thepresent invention comprises substrates labelled or conjugated with thefluorescein dyes of the invention, i.e. structure I. Substrates can bevirtually any molecule or substance to which the dyes of the inventioncan be conjugated, including by way of example and not limitation,proteins, polypeptides, polysaccharides, nucleosides, nucleotides,polynucleotides, lipids, solid supports, organic and inorganic polymers,and combinations and assemblages thereof, such as chromosomes, nuclei,living cells (e.g., bacteria or other microorganisms, mammalian cells,tissues, etc.), and the like. The dyes are conjugated with the substratevia an optional linker by a variety of means, including hydrophobicattraction, ionic attraction, and covalent attachment. Preferably, thedyes are conjugated to the substrate via covalent attachment.

Labelling typically results from mixing an appropriate reactivefluorescent dye and a substrate to be conjugated in a suitable solventin which both are soluble, using methods well-known in the art(Hermanson, Bioconjugate Techniques, Academic Press, San Diego, Calif.pp. 40-55, 643-71, (1996)), followed by separation of the conjugate fromany unconjugated starting materials or unwanted by-products. The dyeconjugate can be stored dry or in solution for later use.

The fluorescein dyes of the invention may include a reactive linkinggroup at one of the substituent positions, R¹-R⁵, R⁷, X¹-X⁵, or covalentattachment of the dye to another molecule, i.e. a substrate. Reactivelinking groups are moieties on the dye and on the substrate which arecapable of forming a covalent bond. Typically the dye has electrophilicfunctional group(s) capable of reacting with nucleophilic functionalgroup(s) on the substrate. Examples of substrate nucleophiles includealcohols, alkoxides, amines, hydroxylamines, and thiols. The selectionof the reactive linking groups used to attach the dye to the substratetypically depends on the complementary functionality on the substrate tobe conjugated. Examples of electrophile reactive linking groups includesuccinimidyl ester, isothiocyanate, sulfonyl chloride, sulfonate ester,silyl halide, 2,6-dichlorotriazinyl, pentafluorophenyl ester,phosphoramidite, maleimide, haloacetyl, epoxide, alkylhalide, allylhalide, aldehyde, ketone, acylazide, anhydride, and iodoacetamide. Asingle type of reactive linking group may be available on the substrate(typical for polysaccharides), or a variety of groups may occur (e.g.amines, thiols, alcohols, phenols), as is typical for proteins. Aconjugated substrate may be conjugated to more than one dye, which maybe the same or different, or to a substrate that is additionallymodified by a hapten. Although some selectively can be obtained bycareful control of the reaction conditions, selectively of labelling isbest obtained by selection of an appropriate reactive dye.

A preferred reactive linking group is N-hydroxysuccinimidyl ester (NHS)of a carboxyl group substituent of the fluorescein dye. The NHS esterform of the dye is a preferred labelling reagent. The NHS ester of thedye may be preformed, isolated, purified, and/or characterized, or itmay be formed in situ and reacted with a nucleophilic group of asubstrate, such as an oligonucleotide, a nucleotide, a peptide, or thelike. Typically, the carboxyl form of the dyes of the present invention,e.g. the dye compounds of Table 1, are reacted with a carbodiimidereagent, e.g. dicyclohexylcarbodiimide, diisopropylcarbodiimide, or auronium reagent, e.g. HBTU(O-benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate)or HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate), and an activator, such as 1-hydroxybenzotriazole(HOBt), and N-hydroxysuccinimide to give the NHS ester of the dye.

Preferred substituent positions for NHS esters on the fluorescein dyesof the invention are X³ and X⁴ (Ia). A representative example of an NHSester is structure Ib:

Another preferred reactive linking group is a phosphoramidite form ofthe dyes of the present invention. Phosphoramidite dye reagents areparticularly useful for the automated synthesis of oligonucleotideslabelled with the dyes of the invention. Oligonucleotides are commonlysynthesized on solid supports by the phosphoramidite method usingphosphoramidite nucleoside reagents (Caruthers, M. and Beaucage, S.“Phosphoramidite compounds and processes”, U.S. Pat. No. 4,415,732,issued Nov. 15, 1983; Caruthers, M. and Matteucci, M. “Process forpreparing polynucleotides”, U.S. Pat. No. 4,458,066, issued Jul. 3,1984; Beaucage, S. and Iyer, R. (1992) “Advances in the synthesis ofoligonucleotides by the phosphoramidite approach”, Tetrahedron48:2223-2311). The phosphoramidite dye reagents can be nucleosidic ornon-nucleosidic, and can be conveniently used to label syntheticpolynucleotides or polynucleotide analogs at their 3′-terminus,5′-terminus and/or at one or more internal positions. Non-nucleosidicforms of phosphoramidite dye reagents have the general structure III

-   -   where DYE is a protected or unprotected form of dye I, including        energy transfer dye. L is a linker. R¹¹ and R¹² taken separately        are alkyl (C₁-C₁₂), alkene, aryl, and cycloalkyl containing up        to 10 carbon atoms, or R¹¹ and R¹² taken together with the        nitrogen atom form a saturated nitrogen heterocycle. R¹³ is a        phosphite ester protecting group which prevents unwanted        extension of the oligonucleotide. Generally, R¹³ is stable to        oligonucleotide synthesis conditions yet is able to be removed        from a synthetic oligonucleotide product with a reagent that        does not adversely affect the integrity of the oligonucleotide        or the dye. A variety of phosphite ester groups having these        characteristics are well-known in the art. Preferably, R¹³ is        methyl; 2-cyanoethyl, —CH₂CH₂CN; and 2-(4-nitrophenyl) ethyl,        —CH₂CH₂(p-NO₂Ph). Preferred embodiments of phosphoramidite dye        reagents are where: (i) R¹¹ and R¹² are each isopropyl, (ii) R¹¹        and R¹² taken together is morpholino, (iii) L is alkyl        (C₁-C₁₂), (iv) R¹³ is 2-cyanoethyl, and (v) DYE is attached at        R⁶ by a linker. Phosphoramidite dye reagents III effect        labelling of a substrate with a single fluorescent dye of the        invention. Where the substrate is an oligonucleotide, the dye        will be attached at the 5′ terminus of the oligonucleotide, as a        consequence of the 3′ to 5′ direction of synthesis. Other        phosphoramidite dye reagents, nucleosidic and non-nucleosidic        allow for labelling at other sites of an oligonucleotide, e.g.        3′ terminus, nucleobase, internucleotide linkage, sugar.        Labelling at the nucleobase, internucleotide linkage, and sugar        sites allows for internal and multiple labelling with        fluorescent dyes.

When reacted with a hydroxyl group, e.g. 5′ terminal OH of anoligonucleotide bound to a solid support, and under mild acidactivation, the phosphoramidite dye reagent III reacts to form aninternucleotide phosphite group which is then oxidized to aninternucleotide phosphate group. In some instances, the dye may containfunctional groups, e.g. phenolic oxygens as in structure I, that requireprotection either during the synthesis of the phosphoramidite dyereagent or during its subsequent use to label molecules such asoligonucleotides. The protecting group(s) used will depend upon thenature of the functional groups, and will be apparent to those havingskill in the art (Greene, T. and Wuts, P. Protective Groups in OrganicSynthesis, 2nd Ed., John Wiley & Sons, New York, 1991). Generally, theprotecting groups used should be stable under the acidic conditions(e.g. trichloroacetic acid, dichloroacetic acid) commonly employed inoligonucleotide synthesis to remove 5′-hydroxyl protecting groups (e.g.,dimethoxytrityl) and labile under the basic conditions (ammoniumhydroxide, aqueous methylamine) used to deprotect and/or cleavesynthetic oligonucleotides from solid supports.

Stable phosphoramidite dye reagents may be formed by initial protectionof the xanthene ring oxygens of structure I, typically esterification,e.g. acylation as pivalate (R=tBu) or benzoate (R=Ph) esters.Esterification causes lactonization of structures Ia which renders thereagent in the nonfluorescent, protected state, e.g. structure Ic:

Where one of X³ and X⁴ is hydrogen and the other is carboxyl, the dyemay be converted to a non-nucleosidic, phosphoramidite dye labellingreagent, e.g. Id, by known reactions, such as activation of thecarboxyl, amidation with 6-amino, 1-hexanol and phosphitylation ofhydroxyl with bis(diisopropylamino)cyanoethylphosphite (Theisen, etal(1992) “Fluorescent dye phosphoramidite labelling of oligonucleotides”,in Nucleic Acid Symposium Series No. 27, Oxford University Press,Oxford, pp. 99-100).

Dyes of the invention may also be covalently attached to solid supportsfor the automated synthesis of oligonucleotides (Mullah, B. and Andrus,A. “Solid support reagents for the direct synthesis of 3′-labelledpolynucleotides”, U.S. Pat. No. 5,736,626, issued Apr. 7, 1998;Caruthers, M. and Matteucci, M. “Process for preparing polynucleotides”,U.S. Pat. No. 4,458,066, issued Jul. 3, 1984). In one embodiment, thedye is attached to a linker that has an attachment to a solid support,controlled-pore-glass (Nelson, etal (1992) “Oligonucleotide labelingmethods 3. Direct labeling of oligonucleotides employing a novel,non-nucleosidic, 2-aminobutyl-1,3-propanediol backbone”, Nucl. AcidsRes. 20:6253-59; Nelson, P. “Multifunctional controlled pore glassreagent for solid phase oligonucleotide synthesis”, U.S. Pat. No.5,141,813, issued Aug. 25, 1992) or polystyrene (Andrus, etal “Automatedsystem for polynucleotide synthesis and purification”, U.S. Pat. No.5,047,524, issued Sep. 10, 1991 and U.S. Pat. No. 5,262,530, issued Nov.16, 1993), and acid-labile functionality for extension withphosphoramidite nucleoside reagents. After cleavage and deprotection,the labelled oligonucleotide may bear a dye of the present invention atthe 3′ terminus.

Nucleotide Labelling

A preferred class of labelled substrates include conjugates ofnucleosides and nucleotides that are labelled with the fluorescein dyesof the invention. Such labelled nucleosides and nucleotides areparticularly useful for labelling polynucleotides formed by enzymaticsynthesis, e.g., labelled nucleotide 5′-triphosphates used in thecontext of PCR amplification, Sanger-type polynucleotide sequencing, andnick-translation reactions.

The dye may be conjugated to either the sugar or nucleobase. In apreferred embodiment, the compound is a terminatingribonucleoside-5′-triphosphate, “terminator”, in which the dye iscovalently attached to the nucleobase moiety. When used in conjunctionwith 2′-deoxyribonucleoside-5′-triphosphates, “dNTP” orribonucleoside-5′-triphosphates, “NTP”, appropriate polymerase enzymesand primed target polynucleotides, such terminators can be used togenerate series of terminated electron deficient nitrogenheterocycle-substituted fluorescein dye labelled polynucleotidefragments via target-mediated enzymatic synthesis for applications suchas DNA sequencing. Nucleosides and nucleotides can be labelled at siteson the sugar or nucleobase moieties. Preferred nucleobase labellingsites include the 8-C of a purine nucleobase, the 7-C or 8-C of a7-deazapurine nucleobase, and the 5-position of a pyrimidine nucleobase.Between a nucleoside or nucleotide and a dye, a linker may attach to adye at any one of positions R¹-R⁷.

The labelled nucleoside or nucleotide may be enzymaticallyincorporatable and enzymatically extendable. Nucleosides or nucleotideslabelled with dyes of the present invention may have structure IV:

-   -   wherein DYE is a protected or unprotected form of dye I,        including energy transfer dye. B is a nucleobase, e.g. uracil,        thymine, cytosine, adenine, 7-deazaadenine, guanine, and        8-deazaguanosine. R⁸ is H, monophosphate, diphosphate,        triphosphate, thiophosphate, or phosphate analog. R⁹ and R¹⁰,        when taken alone, are each independently H, HO, F. Where the        labelled nucleoside or nucleotide is a terminator, R⁹ and R¹⁰        are selected to block polymerase-mediated target-directed        polymerization. In terminator nucleotides, R⁹ and R¹⁰, when        taken alone, are each independently H, F, and a moiety which        blocks polymerase-mediated target-directed polymerization, or        when taken together form 2′-3′-didehydroribose.

Linker L may be:

wherein n is 0, 1, or 2.

Oligonucleotide Labelling

Another preferred class of labelled substrates include conjugates ofoligonucleotides that are labelled with the fluorescein dyes of theinvention. Such labelled polynucleotides or analogs are useful in anumber of important contexts, including as DNA sequencing primers, PCRprimers, oligonucleotide hybridization probes, oligonucleotide ligationprobes, double-labelled 5′-exonuclease (TaqMan™) probes, and the like(Fung, etal. “Amino-derivatized phosphite and phosphate linking agents,phosphoramidite precursors, and useful conjugates thereof”, U.S. Pat.No. 4,757,141, issued Jul. 12, 1988; Andrus, A. “Chemical methods for 5′non-isotopic labelling of PCR probes and primers” (1995) in PCR 2: APractical Approach, Oxford University Press, Oxford, pp. 39-54;Hermanson, (1996) Bioconjugate Techniques, Academic Press, San Diego,Calif. pp. 40-55, 643-71). A labelled oligonucleotide may have structureV:

-   -   where the oligonucleotide comprises 2 to 100 nucleotides. DYE is        a fluorescent dye I, including energy transfer dye. B is a        nucleobase, e.g. uracil, thymine, cytosine, adenine,        7-deazaadenine, guanine, and 8-deazaguanosine. L is a linker.        R¹⁰ is H, OH, halide, azide, amine, alkylamine, alkyl (C¹-C₆),        allyl, alkoxy (C₁-C₆), OCH₃, or OCH₂CH═CH₂. R¹⁵ is H, phosphate,        internucleotide phosphodiester, or internucleotide analog. R¹⁶        is H, phosphate, internucleotide phosphodiester, or        internucleotide analog. In this embodiment, structure V, the        nucleobase-labelled oligonucleotide may bear multiple dyes of        the invention attached through the nucleobases.

Nucleobase-labelled oligonucleotide V may be formed by: (i) enzymaticincorporation of enzymatically incorporatable nucleotide reagents IVwhere R⁸ is triphosphate, by a DNA polymerase or ligase, and (ii)coupling of phosphoramidite dye nucleoside reagent VI by automatedsynthesis.

Generally, if the labelled oligonucleotide is made by enzymaticsynthesis, the following procedure may be used. A target DNA isdenatured and an oligonucleotide primer is annealed to the target DNA. Amixture of enzymatically-incorporatable nucleotides or nucleotideanalogs capable of supporting continuous target-directed enzymaticextension of the primed target (e.g., a mixture including dGTP, dATP,dCTP and dTTP or dUTP) is added to the primed target. At least afraction of the nucleotides is labelled with a dye I or are labelledterminators, as described above. Next, a polymerase enzyme is added tothe mixture under conditions where the polymerase enzyme is active. Alabelled oligonucleotide is formed by the incorporation of the labellednucleotides or terminators during polymerase-mediated strand synthesis.

In an alternative enzymatic synthesis method, two primers are usedinstead of one: one complementary to the (+) strand of the targetsequence and another complementary to the (−) strand of the targetsequence, the polymerase is a thermostable polymerase and the reactiontemperature is cycled between a denaturation temperature and anextension temperature, thereby exponentially amplifying a labelledcomplement to the target sequence by PCR (Innis, etal (1990) PCRProtocols, Eds., Academic Press). One or both primers may be labelledwith a dye of the invention. Alternatively, one or more of thenucleotide 5′-triphosphates may be labelled with a dye of the invention.Each labelling scheme results in a DNA amplification product bearing oneor more dyes of the invention.

Internal labelling of oligonucleotides with fluorescent dyes of theinventions can be conducted with nucleoside phosphoramidite dye reagentsof general structure VI:

-   -   where DYE is a protected or unprotected form of dye I, The        exocyclic amines and other functionalities of nucleobase B may        require protection during the synthesis of the nucleoside        phosphoramidite dye reagent and/or during its subsequent use to        synthesize labelled oligonucleotides. The particular protecting        group(s) selected will depend on the identity of the nucleobase        or nucleobase analog, and will be apparent to those of skill in        the art. For example, the exocyclic amines of adenine and        cytosine can be protected with benzoyl (bz) and the exocyclic        amine of guanine can be protected with dimethylformamide (dmf)        or isobutyryl (ibu) using conventional procedures. Preferably,        the nucleobase is protected with groups that are readily removed        under mild basic conditions. For example, oligonucleotides        synthesized with dA^(bz), dC^(bz), dG^(dmf) and T        phosphoramidite nucleosides (and their corresponding 3′        nucleoside solid supports) can be cleaved and deprotected in 60        minutes in concentrated ammonium hydroxide at 65° C.        (Beaucage, S. and Iyer, R. (1992) “Advances in the synthesis of        oligonucleotides by the phosphoramidite approach”, Tetrahedron        48:2223-2311).

R¹¹ and R¹² taken separately are selected from the group consisting ofalkyl (C₁-C₆), alkene, aryl, and cycloalkyl containing up to 10 carbonatoms, e.g. isopropyl, or R¹¹ and R¹² taken together with the nitrogenatom form a saturated nitrogen heterocycle, e.g. morpholino, R¹³ is aphosphite ester protecting group, e.g. methyl, 2-cyanoethyl, and2-(4-nitrophenyl)ethyl. R¹⁴ is an acid-cleavable hydroxyl protectinggroup, e.g. dimethoxytrityl, which allows subsequent monomer coupling.

The linker L of VI may be

wherein n ranges from 2 to 10;

wherein n is 0, 1, or 2; and

wherein n ranges from 1 to 10.

Reagents IV and VI are effective in preparing oligonucleotides Vlabelled with the dyes of the invention I. Another embodiment of alabelled oligonucleotide is 5′ terminus labelled according to structureVII:

-   -   where X is O, NH, or S, L is alkyldiyl (C₁-C₁₂) or        mobility-modifier, and the labelled oligonucleotide bears only        one DYE. Mobility-modifier linkers affect the electrophoretic        mobility or hydrophobic properties of the oligonucleotide.        Examples of mobility-modifier linkers include ethyleneoxy units,        —(CH₂CH₂O)_(n)—, where n maybe 1 to 100 (Grossman, etal “Method        of DNA sequencing employing a mixed DNA-polymer chain probe”,        U.S. Pat. No. 5,624,800, Issued Apr. 29, 1997). Preferably, n is        from 2 to 20. Labelled oligonucleotide VII may be formed by        automated synthesis with phosphoramidite reagents III, in        particular for example Id. Alternatively, labelled        oligonucleotides VII may be formed by reacting a reactive        linking group form of a dye of the present invention, e.g. Ib,        with a 5′-aminoalkyl oligonucleotide.

In one preferred post-synthesis chemical labelling method anoligonucleotide is labelled as follows. A dye according to structure Ibis dissolved or suspended in DMSO and added in excess (10-20×) to a5′-aminohexyl oligonucleotide in 0.25 M bicarbonate/carbonate buffer atabout pH 9 and allowed to react for 6 hours, e.g. U.S. Pat. No.4,757,141. The dye labelled oligonucleotide VII can be separated fromunreacted dye by passage through a size-exclusion chromatography columneluting with buffer, e.g., 0.1 molar triethylamine acetate (TEAA). Thefraction containing the crude labelled oligonucleotide VII, where L is—(CH₂)₆—, is further purified by reverse phase HPLC employing gradientelution.

Kits

The invention comprises kits comprising the electron-deficient nitrogenheterocycle-substituted fluorescein dyes of the invention and/or theirlabelled conjugates. In one embodiment, the kits are useful forconjugating the dyes of the invention to other molecules, i.e.substrates. Such kits generally comprise a dye of the inventionincluding an optional linking moiety and reagents, enzymes, buffers,solvents, etc. suitable for conjugating the dye to another molecule orsubstance.

In one embodiment, the kits are useful for labelling enzymaticallysynthesized oligonucleotides and polynucleotides with the dyes of theinvention. Such kits generally comprise a labelledenzymatically-incorporatable nucleotide or nucleotide analog accordingto the invention, a mixture of enzymatically-incorporatable nucleotidesor nucleotide analogs capable of supporting continuous primer extensionand a polymerase enzyme. Preferably, the labelledenzymatically-incorporatable nucleotide or nucleotide analog is acompound according to structure IV, most preferably a labelledterminator. Preferred polymerases are thermostable, such as AMPLITAQ®DNA polymerase FA (PE Biosystems, Foster City, Calif.).

In another embodiment, the kits are useful for labelling syntheticoligonucleotides with the phosphoramidite dye reagents of the invention.Such kits generally comprise a phosphoramidite dye reagent, othersynthesis reagents, and/or solid supports optionally for carrying outoligonucleotide synthesis (Andrus, etal “Automated system forpolynucleotide synthesis and purification” U.S. Pat. No. 5,262,530,issued Nov. 16, 1993).

Methods Using Labelled Reagents

The dyes and reagents of the present invention are well suited to anymethod utilizing fluorescent detection, particularly methods requiringthe simultaneous detection of multiple spatially-overlapping analytes(Menchen, etal “4,7-dichlorofluorescein dyes as molecular probes”, U.S.Pat. No. 5,188,934, issued Feb. 23, 1993. Dyes and reagents of theinvention are particularly well suited for identifying classes ofpolynucleotides that have been subjected to a biochemical separationprocedure, such as electrophoresis, or that have been distributed amonglocations in a spatially-addressable hybridization array.

These applications include use of the labelled oligonucleotides as5′-labelled sequencing primers, 5′-labelled polymerase chain reaction(PCR) primers, hybridization probes, and ligation assay probes. PCRapplications include the use of labelled oligonucleotides for genotypingby variable number tandem repeat (VNTR), short tandem repeat (STR), andmicrosatellite methods of amplification of repeat regions ofdouble-stranded DNA that contain adjacent multiple copies of aparticular sequence, with the number of repeating units being variable.Preferably, in such PCR genotyping methods, the PCR primer is labelledwith a dye of the invention.

In a particularly preferred embodiment, the fluorescein dyes of theinvention may be used in quantitative methods and reagents that providereal time or end-point measurements of amplification products during PCR(Gelfand, etal. “Homogeneous assay system using the nuclease activity ofa nucleic acid polymerase”, U.S. Pat. No. 5,210,015, issued May 9, 1993;Livak, etal “Method for Detecting Nucleic Acid Amplification UsingSelf-Quenching Fluorescence Probe”, U.S. Pat. No. 5,538,848, issued Jul.23, 1996). The exonuclease assay (Taqman®) employing fluorescentdye-quencher probes (Livak, etal “Self-quenching fluorescence probe”,U.S. Pat. No. 5,723,591, issued Mar. 3, 1998; Mullah, etal (1998)“Efficient synthesis of double dye-labelled oligodeoxyribonucleotideprobes and their application in a real time PCR assay”, Nucl. Acids Res.26:1026-1031) gives direct detection of polymerase chain reaction (PCR)products in a closed-tube system, with no sample processing beyond thatrequired to perform the PCR. In the Taqman assay, the polymerase thatconducts primer extension and amplifies the polynucleotide alsodisplaces and cleaves a probe annealed to target sequence by 5′ to 3′exonuclease activity. In a Taqman-type assay, the probe isself-quenching, labelled with fluorescent dye and quencher moieties,either of which may be dyes of the invention. Spectral overlap allowsfor efficient energy transfer (FRET) when the probe is intact (Clegg, R.(1992) “Fluorescence resonance energy transfer and nucleic acids”, Meth.Enzymol. 211:353-388). When hybridized to a target sequence, the probeis cleaved during PCR to release a fluorescent signal that isproportional to the amount of target-probe hybrid present (Livak, etal“Method for Detecting Nucleic Acid Amplification Using Self-QuenchingFluorescence Probe”, U.S. Pat. No. 5,538,848, issued Jul. 23, 1996;Livak, etal “Self-quenching fluorescence probe”, U.S. Pat. No.5,723,591, issued Mar. 3, 1998).

In yet another aspect, the invention provides methods of using theelectron deficient nitrogen heterocycle-substituted fluorescein dyes ofthe invention to sequence a target polynucleotide. The method generallycomprises forming a series of differently-sized polynucleotides labelledwith a dye of the invention, separating the series of differently-sizedlabelled polynucleotides based on size and detecting the separatedlabelled polynucleotides based on the fluorescence of the dye.

The series of differently-sized labelled polynucleotides can beconveniently generated by enzymatically extending a primed targetsequence according to well-known methods (Sanger, etal, (1977) “DNAsequencing with chain-terminating inhibitors”, Proc. Natl. Acad. Sci.USA 74:5463-5467; Hunkapiller, etal “Real time scanning electrophoresisapparatus for DNA sequencing”, U.S. Pat. No. 4,811,218, issued Mar. 7,1989; PE Corp., Jan. 1995, ABI PRISM® 377 DNA Sequencer User's Manual,Rev. A, Chapter 2 (P/N 903433, PE Corporation, Foster City, Calif.). Forexample, the series of labelled polynucleotides can be obtained using alabelled primer and enzymatically extending target sequence primed withthe labelled target in the presence of a polymerase, dNTP, and at leastone terminator (e.g., 2′,3′-dideoxyribonucleoside-5′-triphosphate).Alternatively, the series of labelled polynucleotides can be obtained byenzymatically extending an unlabelled primed target in the presence of apolymerase, dNTP and at least one labelled terminator (Bergot, etal“Spectrally resolvable rhodamine dyes for nucleic acid sequencedetermination”, U.S. Pat. No. 5,366,860, issued Nov. 22, 1994). Ineither embodiment, the polymerase serves to extend the primer with dNTPuntil a terminator is incorporated, which terminates the extensionreaction. Once terminated, the series of labelled polynucleotides areseparated based on size and the separated polynucleotides are detectedbased on the fluorescence of the dye labels.

In a particularly advantageous embodiment of this method, four differentfluorescently labelled terminators are used, where each nucleoside islabelled with a different spectrally-resolvable fluorophore, and atleast one of the fluorophores is an electron deficient nitrogenheterocycle-substituted fluorescein dye according to the invention suchthat the set of fluorophores are “mobility matched.” According to thisembodiment, the primed target sequence is enzymatically extended in thepresence of a polymerase, dNTP and the four different fluorescentlylabelled terminators. Following separation based on size, a series ofseparated labelled polynucleotides is obtained in which the emissionproperties of the fluorescent dye reveal the identity of the 3′-terminalnucleotide. In a particularly preferred embodiment, all of thefluorescent dyes of the mobility matched set are excitable using asingle light source.

In a preferred category of methods referred to herein as “fragmentanalysis” or “genetic analysis” methods, labelled polynucleotidesequences, or “fragments” are generated through target-directedenzymatic synthesis using labelled primers or nucleotides, e.g. byligation or polymerase-directed primer extension; the fragments aresubjected to a size-dependent separation process, e.g., electrophoresisor chromatography; and the separated fragments are detected subsequentto the separation, e.g., by laser-induced fluorescence (Hunkapiller,etal “Real time scanning electrophoresis apparatus for DNA sequencing”,U.S. Pat. No. 4,811,218, issued Mar. 7, 1989). In a particularlypreferred embodiment, multiple classes of polynucleotides are separatedsimultaneously and the different classes are distinguished by spectrallyresolvable labels.

In a particularly preferred fragment analysis method, fragments labelledwith dyes of the invention are identified by relative size.Correspondence between fragment size and sequence is established byincorporation of the four possible terminating bases (“terminators”) andthe members of a set of spectrally resolvable dyes. Such sets arereadily assembled from the dyes of the invention by measuring emissionand absorption bandwidths with commercially availablespectrophotometers. Preferably, the chain termination methods of DNAsequencing, i.e. dideoxy DNA sequencing, or Sanger-type sequencing, andfragment analysis is employed. Each of the terminators bears a differentfluorescent dye and collectively the terminators of the experiment beara set of dyes including one or more from the dyes of the invention.

Spectrally-resolvable fluorescent dyes of the invention are also usefulin genotyping experiments after PCR amplification of target. Inparticular, a set of primer oligonucleotides, labelled at the 5′terminus, each with different dyes, can amplify multiple loci anddiscriminate single nucleotide polymorphisms (SNP). Electrophoreticseparation of the dye labelled amplification products, with sizestandards, establishes a profile or characteristic data set indicating acertain genotype dependent on the set of primer sequences.

The covalent joining of polynucleotide probes by ligase enzymes is oneof the most useful tools available to molecular biologists. When twoprobes are annealed to a target sequence where the two probes areadjacent and without intervening gaps, a phosphodiester bond can beformed between a 5′ terminus of one probe and the 3′ terminus of theother probe by a ligase enzyme, (Whiteley, etal “Detection of specificsequences in nucleic acids”, U.S. Pat. No. 4,883,750, issued 1989;Landegren, etal. (1988) “A ligase mediated gene detection technique”,Science 241:1077-80; Nickerson, etal (1990) “Automated DNA diagnosticsusing an ELISA-based oligonucleotide assay” Proc. Natl. Acad. Sci USA87:8923-27). Oligonucleotide ligation assays detect the presence ofspecific sequences in a target sample. Where one or both probes arelabelled with a dye of the invention, the ligation product may bedetected by fluorescence (Grossman, etal (1994) “High-density multiplexdetection of nucleic acid sequences: oligonucleotide ligation assay andsequence-coded separation”, Nucl. Acids Res. 22:4527-34).

Sanger-type sequencing involves the synthesis of a DNA strand by a DNApolymerase in vitro using a single-stranded or double-stranded DNAtarget whose sequence is to be determined. Synthesis is initiated at adefined site based on where an oligonucleotide primer anneals to thetarget. The synthesis reaction is terminated by incorporation of anucleotide analog that will not support continued DNA elongation.Exemplary chain-terminating nucleotide analogs include the2′,3′-dideoxynucleoside 5′-triphosphates (ddNTP) which lack the 3′-OHgroup necessary for 3′ to 5′ DNA chain elongation. When properproportions of dNTP (2′-deoxynucleoside 5′-triphosphates) and one of thefour ddNTP are used, enzyme-catalyzed polymerization will be terminatedin a fraction of the population of chains at each site where the ddNTPis incorporated. If fluorescent dye-labelled primers or labelled ddNTPare used for each reaction, the sequence information can be detected byfluorescence after separation by high-resolution electrophoresis. In thechain termination method, dyes of the invention can be attached toeither sequencing primers or dideoxynucleotides. Dyes can be linked to acomplementary functionality on the 5′ terminus of the primer (Fung, etal“Amino-derivatized phosphite and phosphate linking agents,phosphoramidite precursors, and useful conjugates thereof”, U.S. Pat.No. 4,757,141, issued Jul. 12, 1988), on the nucleobase of a primer; oron the nucleobase of a dideoxynucleotide, e.g. via alkynylamino linkinggroups (Khan, etal “Substituted propargylethoxyamido nucleosides,oligonucleotides and methods for using same”, U.S. Pat. No. 5,770,716,issued Jun. 23, 1998, and U.S. Pat. No. 5,821,356, issued Oct. 13, 1998;Hobbs, F. and Trainor, G. “Alkynylamino-nucleotides”, U.S. Pat. No.5,151,507, issued Sep. 29, 1992).

A typical sequencing test is illustrated in FIG. 16 where a 3′-fluoroterminator is labelled with dye compound 13 through apropargylethoxyamido linker (EO): 3′FddGTP-EO-13:

In the above fragment analysis methods, labelled polynucleotides areseparated by chromatographic, affinity, or electrophoretic procedures.Preferably the separation is electrophoresis (Rickwood and Harnes, Eds.,Gel Electrophoresis of Nucleic Acids: A Practical Approach, IRL PressLimited, London, 1981). The preferred type of electrophoretic matrix iscrosslinked or uncrosslinked polyacrylamide, or other amide-containingpolymer, having a concentration (weight to volume) of between about 2-20weight percent (Madabhushi, etal “Polymers for separation ofbiomolecules by capillary electrophoresis”, U.S. Pat. No. 5,552,028,Issued Sep. 3, 1996). The electrophoretic matrix maybe configured in aslab gel or capillary format (Mathies, etal “Capillary array confocalfluorescence scanner and method”, U.S. Pat. No. 5,274,240, issued Dec.28, 1993). More preferably, the polyacrylamide concentration is betweenabout 4-8 percent. Preferably in the context of DNA sequencing inparticular, the electrophoresis matrix includes a denaturing agent,e.g., urea, formamide, and the like (Maniatis etal, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York, pgs. 179-185(1982); PE Corp., ABI PRISM® 377 DNA Sequencer User's Manual, Rev. A,Chapter 2 (P/N 903433, PE Corporation, Foster City, Calif.) January1995). The optimal electrophoresis conditions, e.g., polymer, polymerconcentration, pH, temperature, concentration of denaturing agent,employed in a particular separation depends on many factors, includingthe size range of the polynucleotides to be separated, their basecompositions, whether they are single stranded or double stranded, andthe nature of the classes for which information is sought byelectrophoresis (Grossman, P. “High resolution DNA sequencing methodusing low viscosity medium”, U.S. Pat. No. 5,374,527, issued Dec. 20,1994). Accordingly application of the invention may require standardpreliminary testing to optimize conditions for particular separations.

Polynucleotides labelled with the dyes of the present invention may alsobe labelled with moieties that affect the rate of electrophoreticmigration, i.e. mobility-modifying labels. Mobility-modifying labelsinclude polymers of ethyleneoxy units, —(CH₂CH₂O)_(n)— where n may be 1to 100 (Grossman, etal “Method of DNA sequencing employing a mixedDNA-polymer chain probe”, U.S. Pat. No. 5,624,800, Issued Apr. 29,1997). Preferably, n is from 2 to 20. Specifically labelling fluoresceindye labelled polynucleotides with additional labels of polyethyleneoxyof discrete and known size allows for another dimension of separation byelectrophoresis and detection, independent of the number of nucleotidesin the polynucleotide. That is, polynucleotides of the same length maybe discriminated upon the bases of spectrally resolvable dye labels andmobility-modifying labels. Polynucleotides bearing both dye labels andmobility-modifying labels may be formed enzymatically by ligation orpolymerase extension of the single-labelled polynucleotide or nucleotideconstituents. Alternatively, synthetic oligonucleotides may bear labelsof dyes of the present invention and mobility-modifiers which areincorporated during automated synthesis, e.g. phosphoramidite reagents,or via post-synthesis coupling, e.g. NHS-label coupling to amino- orthiol-oligonucleotides.

Subsequent to electrophoretic separation, the dye-polynucleotideconjugates are detected by measuring the fluorescence emission from thedye labelled polynucleotides. To perform such detection, the labelledpolynucleotides are illuminated by standard means, e.g: high intensitymercury vapor lamps, lasers, or the like. Preferably the illuminationmeans is a laser having an illumination beam at a wavelength above about450 nm. More preferably, the dye-polynucleotides are illuminated bylaser light generated by an argon-ion or He-Ne gas laser or asolid-state diode laser. The fluorescence is then detected by alight-sensitive detector, e.g., a photomultiplier tube, a chargedcoupled device, or the like. Exemplary electrophoresis detection systemsare described elsewhere (Hoff, etal “Real-time scanning fluorescenceelectrophoresis apparatus for the analysis of polynucleotide fragments”,U.S. Pat. No. 5,543,026, issued Aug. 6, 1996; Mathies, etal “Capillaryarray confocal fluorescence scanner and method”, U.S. Pat. No.5,274,240, issued Dec. 28, 1993; Hunkapiller, etal “Real time scanningelectrophoresis apparatus for DNA sequencing”, U.S. Pat. No. 4,811,218,issued Mar. 7, 1989).

EXAMPLES

The invention is further illustrated by the following examples, whichare intended to be purely exemplary of the present invention and not tolimit its scope in any way.

Example 1 Synthesis of 1,3-Dimethoxy-2-(Tol-2-yl)-Benzene 11,3-Dimethoxyphen-2-yl boronic acid:

(10 g, 54.95 mmol, Frontier Scientific, Inc.), 2-bromotoluene (18.81 g,110 mmol), glyme (200 ml), and tetrakis(triphenylphosphine) palladium,(Ph₃P)₄Pd^(o) (4 g, 3.46 mmol) were stirred for 15 minutes followed bythe addition of 8 g potassium carbonate in 35 ml of water (ref). Afterrefluxing for 6 hr, the reaction mixture was poured into 800 ml of 1:1mixture of water and ethyl acetate. The organic layer was washed withwater (300 ml×2) and brine (200 ml×1), the solvent was removed, and thecrude product was purified by silica-gel chromatography (hexane, ethylacetate 0% to 10%) yielding 10.5 g (84%) of 1 (FIG. 1).

Example 2 Synthesis of 1,3-Dimethoxy-2-(Tol-2-yl)-4-Bromobenzene 2

1,3-Dimethoxy-2-(tol-2-yl)-benzene 1, (10 g, 43.86 mmol),N-bromosuccinimide (8.26 g, 46.4 mmol), and perchloric acid (70%, 0.5ml) were mixed in dichloromethane (200 ml) and stirred for 4 hrs at roomtemperature. The reaction mixture was quenched with sodium bicarbonatesolution (5%, 100 ml), the dichloromethane layer was washed with water(100 ml), brine (100 ml), and the solvent was removed. The crude productwas purified by silica-gel chromatography eluting with hexane and ethylacetate (0% to 10% ethyl acetate) yielding 10.8 g (80%) of 2.

Example 3 Synthesis of 1,3-Dimethoxy-2-(Tol-2-yl)-4-(Pyrid-3-yl)-Benzene3

Pyridine-3-boronic acid (5.81 g, 47.27 mmol, Frontier Scientific, Inc.),1,3-dimethoxy-2-(tol-2-yl)-4-bromobenzene (2) (10.7 g, 34.8mmol), glyme(200 mL), and tetrakis(triphenylphosphine) palladium (Ph₃P)₄Pd^(o) (4 g,3.46 mmol) were stirred for 15 minutes followed by the addition ofpotassium carbonate (16 g in 70 ml of water). After refluxing for 10 hr,the reaction mixture was poured into 800 ml of 1:1 mixture of water andethyl acetate. The organic layer was washed with water (300 ml×2) andbrine (200 ml), the solvent was removed, and the crude product waspurified by silica-gel chromatography eluting with hexane and ethylacetate (10% to 25% ethyl acetate) yielding 8.8 g (83%) of 3.

Example 4 Synthesis of 2-(Tol-2-yl)-4-(Pyrid-3-yl)-1,3-Dihydroxybenzene4

1,3-Dimethoxy-2-(tol-2-yl)-4-(pyrid-3-yl)-benzene 3 (3.5 g, 11.46 mmol),acetic acid (30 ml), and hydrobromic acid (48%) (15 ml) were refluxedfor 24 hr. After cooling the reaction mixture, most of the reagent wasremoved under reduced pressure; the residual acid was removed by mixingthe concentrate with sodium bicarbonate solution (5%) (100 ml). Theproduct was extracted into ethylacetate (100 ml), washed with water (100ml×2) and brine (100 ml), and the ethyl acetate was evaporated. Thecrude product was purified by silica-gel chromatography eluting withdichloromethane and acetone (0% to 10% acetone) yielding 2.8 g (90%) of4.

Example 5 Synthesis of Ketone 5

2-(Tol-2-yl)-4-(pyrid-3-yl)-1,3-dihydroxybenzene 4 (630 mg, 2.25 mmol),2,5-dichlorotrimellitic anhydride:

(Menchen, etal S. “4, 7-dichlorofluorescein dyes as molecular probes”,U.S. Pat. No. 5,885,778, issued Mar. 23, 1999) (590 mg, 2.25 mmol),nitrobenzene (20 ml), and aluminum chloride in nitrobenzene (12 ml, 1M)were stirred at room temperature for 24 hrs. The reaction mixture waspoured into a mixture of ice-water (50 ml), ethyl acetate (100 ml), andn-butanol (20 ml), followed by addition of 10% HCl (50 ml) to dissolvethe aluminum salts. The organic layer was washed with water (50 ml×2)and brine (50 ml), and the solvent was evaporated to yield the productas a mixture of isomers 5a and 5b. Separation by silica-gel columnchromatography with methanol in dichloromethane, (10% to 30% methanol),and (1%) acetic acid yielded 380 mg (31%) of the desired slow-movingisomer 5b, believed to have the structure shown in FIG. 1.

Example 6 1,3-Dimethoxynaphthalene 6

To 1,3-Dihydroxynaphthalene (15 g, 93.6 mmol) and potassium carbonate(20 g, 144.7 mmol) in acetone (200 ml) was added dimethyl sulfate (21ml, 222 mmol). After stirring over-night the reaction mixture was mixedwith 10% sodium hydroxide (100 ml) and extracted with ethyl acetate (200ml×2) The organic layer was washed with water (100 ml×2) evaporated, andthe residue stirred with ammonium hydroxide (50 ml) for 2 hrs to quenchany unreacted dimethyl sulfate. Product was extracted with ethyl acetate(200 ml) and was washed with water (100 ml×2) and brine (100 ml). Thesolvent was evaporated yielding 15 g (85%) of 6.

Example 7 Bis-(1,3-Dimethoxynaphth-2-yl)-Dimethyltin 7

1,3-Dimethoxynaphthalene (6) (7.1 g, 37.73 mmol) was dissolved inanhydrous THF (60 ml), cooled to −70° C., and tetramethylethylenediamine(0.2 ml) was added followed by n-butyllithium (26 ml, 1.6 M in hexane).After stirring the solution cold for 30 minutes and at ambienttemperature for 1 hr, the solution was cooled back to −20° C., anddimethyltin dichloride, SnMe₂Cl₂, (5.05 g in 20 ml THF) was added. Thereaction mixture was stirred at ambient temperature for 2 hr, pouredinto water (100 ml) and extracted with ethyl acetate (100 ml). Theorganic layer was washed with water (50 ml), brine (50 ml), andevaporated. The residue was crystallized from hexane (50 ml), to yield6.8 g (69 %) of 7.

Example 8 1,3-Dimethoxy-2-bromonaphthalene 8

Bis-(1,3-Dimethoxynaphth-2-yl)-dimethyltin 7 (11 g, 21 mmol) in THF (500ml) was cooled to −30° C., and N-bromosuccinimide (8 g, 45 mmol) wasadded. After stirring at −30° C. for 2 hrs the reaction mixture wasquenched with water (200 ml) and ethyl acetate (200 ml). The organicphase was washed with 10% hydrochloric acid (100 ml), water (100 ml×2),brine (100 ml), and evaporated. The crude product was purified bysilica-gel chromatography, eluting with hexane-ethyl acetate (0% to 10%ethyl acetate), to yield 9 g (80%) of 8.

Example 9 2-(Pyrid-3-yl)-1,3-Dimethoxynaphthalene 9

Pyridine-3-boronic acid (3.94 g 32 mmol, Frontier Scientific, Inc.),1,3-dimethoxy -2-bromonaphthalene 8 (6.6 g, 24.7 mmol), glyme (200 ml),and tetrakis (triphenylphosphine) palladium (Ph₃P)₄Pd (3 g, 2.7 mmol)were stirred for 15 minutes followed by the addition of potassiumcarbonate (11.4 g in 50 mL of water). After refluxing over-night, thereaction mixture was poured into 800 ml of 1:1 water:ethyl acetate. Theorganic layer was washed with water (300 ml×2) and brine (200 ml),solvent was removed, and the crude product was purified by silica-gelchromatography eluting with dichloromethane and acetone (0% to 5%acetone) yielding 5.1 g (84%) of 9.

Example 10 2-(Tol-2-yl)-1,3-Dimethoxynaphthalene 10

This compound was prepared essentially by the same procedure as that inEXAMPLE 9 using 8 and tol-2-yl boronic acid (Frontier Scientific, Inc.).

Example 11 Synthesis of 2-(Pyrid-3-yl)-1,3-Dihydroxynaphthalene 11

This compound was prepared by the same procedure as used in EXAMPLE 4using 2-(pyrid-3-yl)-1,3-dimethoxynaphthalene 9 to yield 11.

Example 12 Synthesis of 2-(Tol-2-yl)-1,3-Dihydoxynaphthalene 12

This compound was prepared by the same procedure as used in EXAMPLE 4using 2-(tol-2-yl)-1,3-dimethoxynaphthalene 10 to yield 12.

Example 13 Synthesis of Dye 13

Ketone 5b (250 mg, 0.45 mmol) and2-(pyrid-3-yl)-1,3-dihydroxynaphthalene 11 (109 mg, 0.45mmol) were mixedwith methanesulfonic acid (7 ml) and stirred at 100 ° C. for 1 hr. Thesolution was cooled to room temperature and then poured into ice-water(50 ml). The dye was extracted into n-butanol (50 ml×2) and washed withwater (20 ml×2). The solvent was evaporated under vacuum and dye waspurified by reverse phase silica-gel chromatography (50% methanol) toyield 103 mg (30%) of 13 (FIG. 3).

Example 14 Synthesis of Dye 14

This dye was prepared by the same procedure as used in EXAMPLE 13 using2-(tol-2-yl)-1,3-dihydoxynaphthalene 12 and 5.

Example 15 Synthesis of Dye 15

This dye was prepared by the same procedure as used in EXAMPLE 13 using2-fluoro-1,3-dihydroxynaphthalene (Benson, etal “Asymmetricbenzoxanthene dyes”, U.S. Pat. No. 5,840,999, issued Nov. 24, 1998) and5.

Example 16 Synthesis of Dye 16

2-(Tol-2-yl)-4-(pyrid-3-yl)-1,3-dihydroxybenzene 4 (52 mg, 0.28 mmol),2,5-dichlorotrimellitic anhydride (Menchen, etal“4,7-dichlorofluorescein dyes as molecular probes”, U.S. Pat. No.5,885,778, issued Mar. 23, 1999), (36.5 mg, 14 mmol), andmethanesulfonic acid, (1 ml), were heated and stirred at 130-135 ° C.The solution was cooled to room temperature and then poured intoice-water (50 ml). The crude dye was extracted with n-butanol (50 ml×2)and the extract was washed with water (20 ml×2). Solvent was evaporatedunder vacuum to yield the crude dye as a mixture of two isomers. Theisomers were separated by preparative thin layer chromatography, (silicagel; dichloromethane:methanol:acetic acid/100:10:2 (v:v:v) mobile phase)to yield 24 mg (30%) of the desired slower moving isomer of 16.

Example 17 Synthesis of 2,3-Dimethoxy-4-(Pyrid-3-yl)Benzene 17

2,4-Dimethoxyphenyl-4-yl boronic acid (0.9 g, 4.97 mmol, FrontierScientific, Inc.), 3-bromopyridine (0.82 g, 5 mmol),tetrakis(triphenylphosphine) palladium (Ph₃P)₄Pd^(o) (0.65 g, 0.56mmol), N,N-dimethylformamide (20 ml), and triethylamine (2.1 ml) weremixed and stirred at 110-120° C. for 16 hrs. The reaction mixture waspoured into 100 ml of 1:1 mixture of water and ethylacetate, and theorganic layer was washed with water (50 ml×2) and brine (50 ml). Afterremoval of solvent the crude product was purified by silica-gelchromatography (0% to 10% acetone in dichloromethane) yielding 0.45 g(42.5%) of 17.

Example 18 Synthesis of 4-(Pyrid-3-yl)-1,3-Dihydroxybenzene 18

This compound was prepared by the same procedure as used in EXAMPLE 4using 2,3-Dimethoxy-4-(Pyrid-3-yl) Benzene 17 to yield 18.

Example 19 Synthesis of Dye 19

This dye was prepared by the same procedure as used in EXAMPLE 16 using4-(Pyrid-3-yl)-1,3-Dihydroxybenzene 18 and 2,5-dichlorotrimelliticanhydride.

Example 20 Synthesis of 2,3-Dimethoxy-4-(Pyrid-2-yl)Benzene 20

This compound was prepared by the same procedure as used in EXAMPLE 17using 2,4-Dimethoxyphenyl-4-yl boronic acid and 2-bromopyridine to yield20.

Example 21 Synthesis of 2,3-Dihydroxy-4-(Pyrid-2-yl)Benzene 21

This compound was prepared by the same procedure as used in EXAMPLE 4using 2,3-dimethoxy-4-(pyrid-2-yl) benzene 20 to yield 21.

Example 22 Synthesis of Dye 22

This compound was prepared by the same procedure as used in EXAMPLE 16using 4-(pyrid-3-yl)-1,3-dihydroxybenzene 21 and 2,5-dichlorotrimelliticanhydride.

Example 23 Synthesis of 1,3-Dimethoxy-4-(Quinon-3-yl) benzene 23

This compound was prepared by the same procedure as that used in EXAMPLE17 using 1,3-dimethoxyphen-4-yl boronic acid and 3-bromoquinoline toyield 23.

Example 24 Synthesis of 1,3-Dihydroxy-4-(Quinon-3-yl)benzene 24

This compound was prepared by the same procedure as used in EXAMPLE 4using 1,3-dimethoxy-4-(quinon-3-yl) benzene 23 to yield 24.

Example 25 Synthesis of Dye 25

This dye was prepared by the same procedure as used in EXAMPLE 16 using1,3-dihydroxy-4-(quinon-3-yl) benzene 24 and 2,5-dichlorotrimelliticanhydride.

Example 26 Synthesis of 1,3-Dimethoxy-4-(Quinon-2-yl) benzene 26

This compound was prepared using the same procedure as that used inEXAMPLE 17 using 1,3-dimethoxyphen-4-yl boronic acid and2-bromoquinoline to yield 26.

Example 27 Synthesis of 1,3-Dihydoxy-4-(Quinon-2-yl)benzene 27

This compound was prepared by the same procedure as used in EXAMPLE 4using 1,3-dimethoxy-4-(quinon-2-yl) benzene 26 to yield 27.

Example 28 Synthesis of Dye 28

This dye was prepared by the same procedure as used in EXAMPLE 16 using1,3-dihydoxy-4-(quinon-2-yl)benzene 27 and 2,5-dichlorotrimelliticanhydride.

Example 29 Synthesis of 1,3-Dimethoxy-2-(Pyrid-3-yl)benzene 29

This compound was prepared by the same procedure as used in EXAMPLE 1using 1,3-dimethoxyphen-2-yl boronic acid and 3-bromopyridine to yield29.

Example 30 Synthesis of 1,3-Dimethoxy-2-(Pyrid-3-yl)-4-bromobenzene 30

This compound was prepared by the same procedure as used in EXAMPLE 2using 1,3-Dimethoxy-2-(Pyrid-3-yl) benzene 29 and N-bromosuccinimide toyield 30.

Example 31 Synthesis of 1,3-Dimethoxy-2-(Pyrid-3-yl)-4-phenylbenzene 31

This compound was prepared by the same procedure as used in EXAMPLE 3using 1,3-dimethoxy-2-(pyrid-3-yl)-4-bromobenzene 30 and phenyl boronicacid to yield 31.

Example 32 Synthesis of 1,3-Dihydroxy-2-(Pyrid-3-yl)-4-phenylbenzene 32

This compound was prepared by the same procedure as used in EXAMPLE 4using 1,3-dimethoxy-2-(pyrid-3-yl)-4-phenylbenzene 31 to yield 32.

Example 33 Synthesis of Dye 33

This dye was prepared by the same procedure as used in EXAMPLE 16 using1,3-dihydroxy-2-(pyrid-3-yl)-4-phenylbenzene 32 and2,5-dichlorotrimellitic anhydride.

Example 34 Synthesis of 1,3-Dimethoxy-2-(Tol-2-yl)-4-Phenylbenzene 34

This compound was prepared by the same procedure as used in EXAMPLE 3using 1,3-dimethoxy-2-(tol-2-yl)-4-bromobenzene 2 and phenyl boronicacid to yield 34.

Example 35 Synthesis of 1,3-Dihydroxy-2-(Tol-2-yl)-4-Phenylbenzene 35

This compound was prepared by the same procedure as used in EXAMPLE 4using 1,3-dimethoxy-2-(tol-2-yl)-4-phenylbenzene 34 to yield 35.

Example 36 Synthesis of Dye 36

This due was prepared by the same procedure as used in EXAMPLE 16 using1,3-dihydroxy-2-(tol-2-yl)-4-phenylbenzene 35 and2,5-dichlorotrimellitic anhydride.

Example 37 Synthesis of 1,3-Dimethoxy-2-(Pyrid-2-yl)benzene 37

This compound was prepared by the same procedure as used in EXAMPLE 1using 1,3-dimethoxyphen-2-yl boronic acid and 2-bromopyridine to yield37.

Example 38 Synthesis of 1,3-Dimethoxy-2-(Pyrid-2-yl)-4-Bromobenzene 38

This compound was prepared by the same procedure as used in EXAMPLE 2using 1,3-Dimethoxy-2-(Pyrid-2-yl) benzene 37 and N-bromosuccinimide toyield 38.

Example 39 Synthesis of1,3-Dimethoxy-2-(Pyrid-2-yl)-4-(Naphth-2-yl)benzene 39

This compound was prepared by the same procedure as used in EXAMPLE 3using 1,3-dimethoxy-2-(pyrid-2-yl)-4-bromobenzene 38 and naphth-2-ylboronic acid to yield 39.

Example 40 Synthesis of1,3-Dihydroxy-2-(Pyrid-2-yl)-4-(Naphth-2-yl)benzene 40

This compound was prepared by the same procedure as used in EXAMPLE 4,demethylating 1,3-Dimethoxy-2-(Pyrid-2-yl)-4-(Naphth-2-yl)benzene 39 toyield 40.

Example 41 Synthesis of Dye 41

This dye was prepared by the same procedure as used in EXAMPLE 16 using1,3-dihydroxy-2-(pyrid-2-yl)-4-(naphth-2-yl)benzene 40 and2,5-dichlorotrimellitic anhydride.

Example 42 Properties of Dyes

Certain properties of some of the fluorescein dyes of the presentinvention were measured, Table 1.

TABLE 1 Dye properties Dye Compound Em. Max. (nm)^(a) FWHM^(b) Rel.Photostability^(c) 13 585 45 0.8 14 583 44 15 591 45 0.25 16 570 36 0.7819 554 37 1.5 22 556 37 2.2 25 562 38 1.6 28 564 39 1.5 33 571 38 1.0 36572 38 1.2 41 575 39 1.1 ^(a)emission maxima of the acid form of the dye^(b)full width, half maximum ^(c)relative to 5-carboxyfluorescein(5-FAM)

All publications and patent applications are herein incorporated byreference to the as if each individual publication or patent applicationwas specifically and indicated to be incorporated by reference.

Although certain embodiments have been described in detail above, thosehaving ordinary skill in the art will clearly understand that manymodifications are possible in the preferred embodiments withoutdeparting from the teachings thereof. All such modifications areintended to be encompassed within the following claims.

1. A compound having the formula:

wherein: at least one of R¹, R², R³, R⁴, R⁵, or R⁷ is anelectron-deficient nitrogen heterocycle; R¹, when taken alone, is H, F,Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆) alkoxy, sulfonate,sulfone, amino, imminium, amido, nitrile, reactive linking group,phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, orwhen taken together with R⁷ is benzo or heterocycle; R², when takenalone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆)alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactivelinking group, phenyl, substituted phenyl, aryl, substituted aryl, orheterocycle; R³, when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆)substituted alkyl, (C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium,amido, nitrile, reactive linking group, phenyl, substituted phenyl,aryl, substituted aryl, or heterocycle; R⁴, when taken alone, is H, F,Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆) alkoxy, sulfonate,sulfone, amino, imminium, amido, nitrile, reactive linking group,phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, orwhen taken together with R⁵ is benzo or heterocycle; R⁵, when takenalone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl, (C₁-C₆)alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile, reactivelinking group, phenyl, substituted phenyl, aryl, substituted aryl, orheterocycle, or when taken together with R⁴ is benzo or heterocycle; R⁷,when taken alone, is H, F, Cl, (C₁-C₆) alkyl, (C₁-C₆) substituted alkyl,(C₁-C₆) alkoxy, sulfonate, sulfone, amino, imminium, amido, nitrile,reactive linking group, phenyl, substituted phenyl, aryl, substitutedaryl, or heterocycle, or when taken together with R¹ is benzo orheterocycle; and R⁶ is selected from the group consisting of (C₁-C₆)alkyl, (C₂-C₆) alkene, (C₂-C₆) alkyne, cyano, heterocyclic aromatic,phenyl, and substituted phenyl having the structure:

wherein X¹, X², X³, X⁴ and X⁵ taken separately are H, Cl, F, (C₁-C₆)alkyl, (C₂-C₆) alkene, (C₂-C₆) alkyne, CO₂H, SO₃H, CH₂OH, or reactivelinking group.
 2. The fluorescein dye of claim 1 wherein theelectron-deficient heterocycle is selected from the group consisting of2-pyridyl, 3-pyridyl, 4-pyridyl, 2-quinolyl, 3-quinolyl, 4-quinolyl,2-imidazole, 4-imidazole, 3-pyrazole, 4-pyrazole, pyridazine,pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline,3-(1,2,4-N)-triazolyl, 5-(1,2,4-N)-triazolyl, 5-tetrazolyl, 4-(1-O,3-N)-oxazole, 5-(1-O, 3-N)-oxazole, 4-(1-S, 3-N)-thiazole, 5-(1-S,3-N)-thiazole, 2-benzoxazole, 2-benzothiazole,4-(1,2,3-N)-benzotriazole, and benzimidazole.
 3. The fluorescein dye ofclaim 1 wherein R⁴ taken together with R⁵ is benzo.
 4. The fluoresceindye of claim 1 wherein R¹ taken together with R⁷ is benzo.
 5. Thefluorescein dye of claim 1 in which R⁶ is a substituted phenyl havingthe structure:


6. The fluorescein dye of claim 5 wherein one of X³ and X⁴ is carboxyland the other is hydrogen.
 7. The fluorescein dye of claim 1 wherein R¹,R², R³ and R⁴ each taken separately are phenyl or substituted phenyl. 8.The fluorescein dye of claim 1 wherein R¹, R², R³ and R⁴ each takenseparately are naphthyl or substituted naphthyl.
 9. The fluorescein dyeof claim 1 wherein R² and R³ each taken separately are fluoro or chloro.10. The fluorescein dye of claim 1 wherein R² and R³ each takenseparately are 2-pyridyl or 3-pyridyl.
 11. The fluorescein dye of claim1 wherein R² and R³ each taken separately are 2-quinolyl or 3-quinolyl.12. The fluorescein dye of claim 1 wherein R⁵ and R⁷ are hydrogen. 13.The fluorescein dye of claim 1 in which the reactive linking group isselected from the group consisting of succinimidyl ester,isothiocyanate, sulfonyl chloride, 2,6-dichlorotriazinyl,pentafluorophenyl ester, phosphoramidite, maleimide, haloacetyl, andiodoacetamide.
 14. Fluorescein dyes having the structures:


15. A method of labelling a substrate with a fluorescein dye of claim 1,comprising the step of reacting the substrate with the reactive linkinggroup of the fluorescein dye wherein a substrate-dye conjugate isformed.
 16. The method of claim 15 wherein the reactive linking group isN-hydroxysuccinimide.
 17. The method of claim 15 wherein the reactivelinking group is phosphoramidite.
 18. The method of claim 15 wherein thesubstrate is selected from the group consisting of a polynucleotide, anucleotide, a nucleoside, a peptide, a protein, a carbohydrate, aligand, a particle, and a surface.
 19. The method of claim 18 whereinthe particle is a nanoparticle, a microsphere, a bead, and a liposome.20. The method of claim 18 wherein the surface is glass.
 21. An energytransfer dye compound comprising: a donor dye capable of absorbing lightat a first wavelength and emitting excitation energy in response linkedby a linker to an acceptor dye capable of absorbing the excitationenergy emitted by the donor dye and fluorescing at a second wavelengthin response; wherein at least one of the donor dye and acceptor dye is afluorescein dye of claim
 1. 22. The energy transfer dye of claim 21wherein the linker has the structure:


23. The energy transfer dye of claim 21 wherein the linker has thestructure:


24. The energy transfer dye of claim 21 in which the linker has thestructure:

wherein n is 1 or
 2. 25. A labelled nucleoside or nucleotide having theformula:

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye ofclaim 23; B is a nucleobase; R⁸ is H, monophosphate, diphosphate,triphosphate, thiophosphate, or phosphate analog; R⁹ and R¹⁰, when takenalone, are each independently H, HO, F, and a moiety which blockspolymerase-mediated target-directed polymerization, or when takentogether form 2′-3′-didehydroribose; and L is a linker.
 26. The labellednucleoside or nucleotide of claim 25 wherein B is selected from thegroup consisting of uracil, thymine, cytosine, adenine, 7-deazaadenine,guanine, and 8-deazaguanosine.
 27. The labelled nucleoside or nucleotideof claim 25 in which L is:

wherein n is 0, 1, or
 2. 28. The labelled nucleoside or nucleotide ofclaim 25 which is enzymatically incorporatable.
 29. The labellednucleoside or nucleotide of claim 25 which is a terminator.
 30. Theterminator nucleotide of claim 29 which has the structure:

wherein DYE is a fluorescein dye; B is a nucleobase; R⁸ is triphosphate,α-thiotriphosphate, or triphosphate analog; R⁹ and R¹⁰, when takenalone, are each independently H, F, and a moiety which blockspolymerase-mediated target-directed polymerization, or when takentogether form 2′-3′-didehydroribose; and L is a linker.
 31. The labellednucleoside or nucleotide of claim 25 which is enzymatically extendable.32. A labelled oligonucleotide having the formula:

wherein the oligonucleotide comprises 2 to 100 nucleotides; DYE is afluorescein dye of claim 1 or an energy transfer dye of claim 21; B is anucleobase; L is a linker; R¹⁰ is H, OH, halide, azide, amine,alkylamine, alkyl (C₁-C₆), allyl, alkoxy (C₁-C₆), OCH₃, or OCH₂CH═CH₂;R¹⁵ is H, phosphate, internucleotide phosphodiester, or internucleotideanalog; and R¹⁶ is H, phosphate, internucleotide phosphodiester, orinternucleotide analog.
 33. A labelled oligonucleotide having theformula:

wherein the oligonucleotide comprises 2 to 100 nucleotides; DYE is afluorescein dye of claim 1; X is O, NH, or S; B is a nucleobase; L is alinker; R¹⁰ is H, OH, halide, azide, amine, alkylamine, alkyl (C₁-C₆),allyl, alkoxy (C₁-C₆), OCH₃, or OCH₂CH═CH₂; and R¹⁵ is internucleotidephosphodiester or internucleotide analog.
 34. The labelledoligonucleotide of claim 33 in which L is alkyldiyl (C₁-C₁₂).
 35. Thelabelled oligonucleotide of claim 33 in which L is a mobility-modifiercomprising —(CH₂CH₂O)_(n)—, where n is 1 to
 100. 36. A phosphoramiditecompound having the formula:

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye ofclaim 21; L is a linker; R¹¹ and R¹² taken separately are selected fromthe group consisting of alkyl (C₁-C₁₂), alkene, aryl, and cycloalkylcontaining up to 10 carbon atoms; or R¹¹ and R¹² taken together with thenitrogen atom form a saturated nitrogen heterocycle; and R¹³ is aphosphite ester protecting group.
 37. The phosphoramidite compound ofclaim 36 wherein R¹³ is selected from the group consisting of methyl,2-cyanoethyl, and 2-(4-nitrophenyl)ethyl.
 38. The phosphoramiditecompound of claim 36 wherein R¹¹ and R¹² are each isopropyl.
 39. Thephosphoramidite compound of claim 36 wherein R¹¹ and R¹² taken togetheris morpholino.
 40. The phosphoramidite compound of claim 36 wherein L isalkyldiyl (C₁-C₁₂).
 41. The phosphoramidite compound of claim 36 whereinthe fluorescein dye is attached at R⁶ by a linker.
 42. Thephosphoramidite compound of the structure:

wherein DYE is a fluorescein dye of claim
 1. 43. A phosphoramiditecompound having the formula

wherein DYE is a fluorescein dye of claim 1 or an energy transfer dye ofclaim 21; B is a nucleobase; L is a linker; R¹¹ and R¹² taken separatelyare selected from the group consisting of alkyl (C₁-C₆), alkene, aryl,and cycloalkyl containing up to 10 carbon atoms; or R¹¹ and R¹² takentogether with the nitrogen atom form a saturated nitrogen heterocycle;R¹³ is a phosphite ester protecting group; and R¹⁴ is an acid-cleavablehydroxyl protecting group.
 44. The phosphoramidite compound of claim 43wherein R¹³ is selected from the group consisting of methyl,2-cyanoethyl, and 2-(4-nitrophenyl)ethyl.
 45. The phosphoramiditecompound of claim 43 wherein R¹¹ and R¹² are each isopropyl.
 46. Thephosphoramidite compound of claim 43 wherein R¹¹ and R¹² taken togetheris morpholino.
 47. The compound of claim 43 wherein L is:

and n ranges from 2 to
 10. 48. The compound of claim 43 wherein L is:

and n is0, 1, or
 2. 49. The compound of claim 43 wherein L is

and n ranges from 1 to
 10. 50. The compound of claim 43 wherein B isselected from the group consisting of uracil, thymine, cytosine,adenine, 7-deazaadenine, guanine, and 8-deazaguanosine.
 51. A method ofsynthesizing a labelled oligonucleotide comprising the step of couplinga phosphoramidite compound of claim 36 to an oligonucleotide on a solidsupport.
 52. A method of synthesizing a labelled oligonucleotidecomprising the step of coupling a nucleoside phosphoramidite reagent toa solid support wherein the solid support is labelled with a dyeaccording to claim 1 or an energy transfer compound of claim
 21. 53. Amethod of generating a labelled primer extension product, comprising thestep of enzymatically extending a primer-target hybrid in the presenceof a mixture of enzymatically-extendable nucleotides capable ofsupporting continuous primer extension and a terminator, wherein saidprimer or said terminator is labelled with a dye according to claim 1 oran energy transfer compound of claim
 21. 54. A method of oligonucleotideligation, comprising the steps of: annealing two probes to a targetsequence and forming a phosphodiester bond between the 5′ terminus ofone probe and the 3′ terminus of the other probe; wherein one or bothprobes are labelled with a dye according to claim 1 or an energytransfer compound of claim
 21. 55. A method of fragment analysiscomprising the steps of: subjecting polynucleotide fragments, thefragments being labelled with a fluorescein dye of claim 1 or an energytransfer compound of claim 21, to a size-dependent separation process;and detecting the labelled polynucleotide fragment subsequent to theseparation process.
 56. The method of claim 55 wherein the fragments arelabelled with a mobility-modifying label.
 57. The method of claim 55wherein the fragments are formed by ligation.
 58. The method of claim 55wherein the size-dependent separation process of electrophoresis and thelabelled polynucleotide fragment is detected by fluorescence.
 59. Amethod of amplification comprising the steps of: annealing two or moreprimers to a target DNA sequence and extending the primers by polymeraseand a mixture of enzymatically-extendable nucleotides; wherein a primeror a nucleotide is labelled with a dye according to claim
 1. 60. Amethod of amplification comprising the steps of: annealing two or moreprimers and a fluorescent dye-quencher probe to a target DNA sequenceand extending the primers by polymerase and a mixture ofenzymatically-extendable nucleotides; wherein the probe is labelled witha dye according to claim
 1. 61. A kit for labelling an oligonucleotide,comprising a dye including a reactive linking group according to claim 1and an oligonucleotide.
 62. A kit for labelling an oligonucleotide,comprising a phosphoramidite compound according to claim 36 and anoligonucleotide.
 63. A kit for generating a labelled primer extensionproduct, comprising enzymatically-extendable nucleotides capable ofsupporting continuous primer extension, a terminator and a primer,wherein said primer or said terminator is labelled with a dye accordingto claim
 1. 64. A kit for generating a labelled primer extensionproduct, comprising enzymatically-extendable nucleotides capable ofsupporting continuous primer extension, a terminator and a primer,wherein said primer or said terminator is labelled with an energytransfer dye of claim
 21. 65. A kit for generating a labelled primerextension product, comprising enzymatically-extendable nucleotidescapable of supporting continuous primer extension, a terminator and aprimer, wherein said primer or said terminator is labelled with a dyeaccording to claim
 14. 66. The kit of claim 65 in which the terminatoris a set of four different terminators, one which terminates at a targetA, one which terminates at a target G, one which terminates at a targetC and one which terminates at a target T or U.
 67. The kit of claim 66in which the set of four different terminators is a set ofmobility-matched terminators.
 68. A compound having a formula:

wherein at least one of R ¹ , R ² , R ³ , R ⁴ , R ⁵ , or R ⁷ is anelectron-deficient nitrogen heterocycle; R ¹ is H, F, Cl, (C ₁ -C ₆)alkyl, (C ₁ -C ₆) substituted alkyl, (C ₁ -C ₆) alkoxy, sulfonate,sulfone, amino, imminium, amido, nitrile, reactive linking group,phenyl, substituted phenyl, aryl, substituted aryl, or heterocycle, orwhen taken together with R ⁷ is benzo or heterocycle; R ² is H, F, Cl,(C ₁ -C ₆) alkyl, (C ₁ -C ₆) substituted alkyl, (C ₁ -C ₆) alkoxy,sulfonate, sulfone, amino, imminium, amido, nitrile, reactive linkinggroup, phenyl, substituted phenyl, aryl, substituted aryl, orheterocycle; R ³ is H, F, Cl, (C ₁ -C ₆) alkyl, (C ₁ -C ₆) substitutedalkyl, (C ₁ -C ₆) alkoxy, sulfonate, sulfone, amino, imminium, amido,nitrile, reactive linking group, phenyl, substituted phenyl, aryl,substituted aryl, or heterocycle; R ⁴ is H, F, Cl, (C ₁ -C ₆) alkyl, (C₁ -C ₆) substituted alkyl, (C ₁ -C ₆) alkoxy, sulfonate, sulfone, amino,imminium, amido, nitrile, reactive linking group, phenyl, substitutedphenyl, aryl, substituted aryl, or heterocycle, or when taken togetherwith R ⁵ is benzo or heterocycle; R ⁵ is H, F, Cl, (C ₁ -C ₆) alkyl, (C₁ -C ₆) substituted alkyl, (C ₁ -C ₆) alkoxy, sulfonate, sulfone, amino,imminium, amido, nitrile, reactive linking group, phenyl, substitutedphenyl, aryl, substituted aryl, or heterocycle, or when taken togetherwith R ⁴ is benzo or heterocycle; R ⁷ is H, F, Cl, (C ₁ -C ₆) alkyl, (C₁ -C ₆) substituted alkyl, (C ₁ -C ₆) alkoxy, sulfonate, sulfone, amino,imminium, amido, nitrile, reactive linking group, phenyl, substitutedphenyl, aryl, substituted aryl, or heterocycle, or when taken togetherwith R ⁵ is benzo or heterocycle; and X ¹ , X ² , X ³ , X ⁴ and X ⁵ areindividually selected from the group consisting of H, Cl, F, (C ₁ -C ₆)alkyl, (C ₂ -C ₆) alkene, (C ₂ -C ₆) alkyne, CO ₂ H, SO ₃ H, CH ₂ OH,and reactive linking group.
 69. The compound of claim 68 wherein atleast one of X¹ , X ² , X ³ , X ⁴ and X ⁵ is SO ₃ H.
 70. The compoundclaim 69 wherein the electron-deficient nitrogen heterocycle is selectedfrom the group consisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2-quinolyl, 3 -quinolyl, 4 -quinolyl, 2 -imidazole, 4 -imidazole, 3-pyrazole, 4 -pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline,pthalazine, quinazoline, quinoxaline, 3 -( 1,2,4 -N)-triazolyl, 5 -(1,2,4 -N)-triazolyl, 5 -tetrazolyl, 4 -( 1 -O, 3 -N)-oxazole, 5 -(1-O, 3-N)-oxazole, 4 -( 1 -S, 3 -N)-thiazole, 5 -( 1 -S, 3 -N)-thiazole, 2-benzoxazole, 2 -benzothiazole, 4 -( 1,2,3 -N)-benzotriazole, andbenzimidazole.
 71. The compound of claim 69 wherein theelectron-deficient nitrogen heterocycle is selected from the groupconsisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2 -quinolyl, 3-quinolyl, and 4 -quinolyl.
 72. The compound of claim 69 wherein each ofR¹ and R ⁴ is an electron-deficient nitrogen heterocycle selected fromthe group consisting of 2 -pyridyl and 3 -pyridyl.
 73. The compound ofclaim 69 wherein each of R¹ and R ⁴ is 2 -pyridyl.
 74. The compound ofclaim 69 wherein each of R¹ and R ⁴ is 3 -pyridyl.
 75. The compound ofclaim 69 wherein at least one of R⁵ and R ⁷ is H.
 76. The compound ofclaim 69 wherein: each of R ¹ and R ⁴ is an electron-deficient nitrogenheterocycle; each of R ² and R ³ is H; and each of R ⁵ and R ⁷ is H. 77.An energy transfer dye compound comprising: a donor dye capable ofabsorbing light at a first wavelength and emitting excitation energy inresponse linked by a linker to an acceptor dye capable of absorbing theexcitation energy emitted by the donor dye and fluorescing at a secondwavelength in response; wherein at least one of the donor dye andacceptor dye is a compound of claim 69 .
 78. The energy transfer dyecompound of claim 77 wherein the electron-deficient nitrogen heterocycleis selected from the group consisting of 2 -pyridyl, 3 -pyridyl, 4-pyridyl, 2 -quinolyl, 3 -quinolyl, 4 -quinolyl, 2 -imidazole, 4-imidazole, 3 -pyrazole, 4 -pyrazole, pyridazine, pyrimidine, pyrazine,cinnoline, pthalazine, quinazoline, quinoxaline, 3 -( 1,2,4-N)-triazolyl, 5 -( 1,2,4 -N)-triazolyl, 5 -tetrazolyl, 4 -( 1 -O, 3-N)-oxazole, 5 -(1-O, 3 -N)-oxazole, 4 -( 1 -S, 3 -N)-thiazole, 5 -( 1-S, 3 -N)-thiazole, 2 -benzoxazole, 2 -benzothiazole, 4 -( 1,2,3-N)-benzotriazole, and benzimidazole.
 79. The energy transfer dyecompound of claim 77 wherein the electron-deficient nitrogen heterocycleis selected from the group consisting of 2 -pyridyl, 3 -pyridyl, 4-pyridyl, 2 -quinolyl, 3 -quinolyl, and 4 -quinolyl.
 80. The energytransfer dye compound of claim 77 wherein each of R¹ and R ⁴ is anelectron-deficient nitrogen heterocycle selected from the groupconsisting of 2 -pyridyl and 3 -pyridyl.
 81. The energy transfer dyecompound of claim 77 wherein each of R¹ and R ⁴ is 2 -pyridyl.
 82. Theenergy transfer dye compound of claim 77 wherein each of R¹ and R ⁴ is 3-pyridyl.
 83. The energy transfer dye compound of claim 77 wherein atleast one of R⁵ and R ⁷ is H.
 84. The energy transfer dye compound ofclaim 77 wherein: each of R ¹ and R ⁴ is an electron-deficient nitrogenheterocycle; each of R ² and R ³ is H; and each of R ⁵ and R ⁷ is H. 85.A labeled nucleoside or nucleotide having a formula:

wherein DYE is a compound of claim 69 or an energy transfer dye compoundof claim 77 in which the linker has a structure:

wherein B is a nucleobase; R ⁸ is H, monophosphate, diphosphate,triphosphate, thiophosphate, or phosphate analog; R ⁹ and R ¹⁰ , whentaken alone, are each independently H, HO, F, or a moiety which blockspolymerase-mediated target-directed polymerization, or when takentogether, form 2′- 3′-didehydroribose; and L is a linker.
 86. Thelabeled nucleoside or nucleotide of claim 85 wherein theelectron-deficient nitrogen heterocycle is selected from the groupconsisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2 -quinolyl, 3-quinolyl, 4 -quinolyl, 2 -imidazole, 4 -imidazole, 3 -pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine,quinazoline, quinoxaline, 3 -( 1,2,4 -N)-triazolyl, 5 -( 1,2,4-N)-triazolyl, 5 -tetrazolyl, 4 -( 1 -O, 3 -N)-oxazole, 5 -( 1 -O, 3-N)-oxazole, 4 -( 1 -S, 3 -N)-thiazole, 5 -( 1 -S, 3 -N)-thiazole, 2-benzoxazole, 2 -benzothiazole, 4 -( 1,2,3 -N)-benzotriazole, andbenzimidazole.
 87. The labeled nucleoside or nucleotide of claim 85wherein the electron-deficient nitrogen heterocycle is selected from thegroup consisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2 -quinolyl, 3-quinolyl, and 4 -quinolyl.
 88. The labeled nucleoside or nucleotide ofclaim 85 wherein each of R¹ and R ⁴ is an electron-deficient nitrogenheterocycle selected from the group consisting of 2 -pyridyl and 3-pyridyl.
 89. The labeled nucleoside or nucleotide of claim 85 whereineach of R¹ and R ⁴ is 2 -pyridyl.
 90. The labeled nucleoside ornucleotide of claim 85 wherein each of R¹ and R ⁴ is 3 -pyridyl.
 91. Thelabeled nucleoside or nucleotide of claim 85 wherein at least one of R⁵and R ⁷ is H.
 92. The labeled nucleoside or nucleotide of claim 85wherein: each of R ¹ and R ⁴ is an electron-deficient nitrogenheterocycle; each of R ² and R ³ is H; and each of R ⁵ and R ⁷ is H. 93.A labelled oligonucleotide having a formula:

wherein the oligonucleotide comprises 2 to 100 nucleotides; DYE is acompound of claim 69 or an energy transfer dye compound of claim 77 ; Bis a nucleobase; L is a linker; R ¹⁰ is H, OH, halide, azide, amine,alkylamine, alkyl (C ₁ -C ₆), allyl, alkoxy (C ₁ -C ₆), OCH ₃ , or OCH ₂CH═CH ₂ ; R ¹⁵ is H, phosphate, internucleotide phosphodiester, orinternucleotide analog; and R ¹⁶ is H, phosphate, internucleotidephosphodiester, or internucleotide analog.
 94. The labelledoligonucleotide of claim 93 wherein the electron-deficient nitrogenheterocycle is selected from the group consisting of 2 -pyridyl, 3-pyridyl, 4 -pyridyl, 2 -quinolyl, 3 -quinolyl, 4 -quinolyl, 2-imidazole, 4 -imidazole, 3 -pyrazole, 4 -pyrazole, pyridazine,pyrimidine, pyrazine, cinnoline, pthalazine, quinazoline, quinoxaline, 3-( 1,2,4 -N)-triazolyl, 5 -( 1,2,4 -N)-triazolyl, 5 -tetrazolyl, 4 -( 1-O, 3 -N)-oxazole, 5 -( 1 -O, 3 -N)-oxazole, 4 -( 1 -S, 3 -N)-thiazole,5 -( 1 -S, 3 -N)-thiazole, 2 -benzoxazole, 2 -benzothiazole, 4 -( 1,2,3-N)-benzotriazole, and benzimidazole.
 95. The labelled oligonucleotideof claim 93 wherein the electron-deficient nitrogen heterocycle isselected from the group consisting of 2 -pyridyl, 3 -pyridyl, 4-pyridyl, 2 -quinolyl, 3 -quinolyl, and 4 -quinolyl.
 96. The labelledoligonucleotide of claim 93 wherein each of R¹ and R ⁴ is anelectron-deficient nitrogen heterocycle selected from the groupconsisting of 2 -pyridyl and 3 -pyridyl.
 97. The labelledoligonucleotide of claim 93 wherein each of R¹ and R ⁴ is 2 -pyridyl.98. The labelled oligonucleotide of claim 93 wherein each of R¹ and R ⁴is 3 -pyridyl.
 99. The labelled oligonucleotide of claim 93 wherein atleast one of R⁵ and R ⁷ is H.
 100. The labelled oligonucleotide of claim93 wherein: each of R ¹ and R ⁴ is an electron-deficient nitrogenheterocycle; each of R ² and R ³ is H; and each of R ⁵ and R ⁷ is H.101. A method of generating a labelled primer extension product,comprising: enzymatically extending a primer-target hybrid in thepresence of a mixture of enzymatically-extendable nucleotides capable ofsupporting continuous primer extension and a terminator, wherein saidprimer or said terminator is labelled with a compound of claim 69 or anenergy transfer dye compound of claim 77 .
 102. The method of claim 101wherein the electron-deficient nitrogen heterocycle is selected from thegroup consisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2 -quinolyl, 3-quinolyl, 4 -quinolyl, 2 -imidazole, 4 -imidazole, 3 -pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine,quinazoline, quinoxaline, 3 -( 1,2,4 -N)-triazolyl, 5 -( 1,2,4-N)-triazolyl, 5 -tetrazolyl, 4 -( 1 -O, 3 -N)-oxazole, 5 -( 1 -O, 3-N)-oxazole, 4 -( 1 -S, 3 -N)-thiazole, 5 -( 1 -S, 3 -N)-thiazole, 2-benzoxazole, 2 -benzothiazole, 4 -( 1,2,3 -N)-benzotriazole, andbenzimidazole.
 103. The method of claim 101 wherein theelectron-deficient nitrogen heterocycle is selected from the groupconsisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2 -quinolyl, 3-quinolyl, and 4 -quinolyl.
 104. The method of claim 101 wherein each ofR¹ and R ⁴ is an electron-deficient nitrogen heterocycle selected fromthe group consisting of 2 -pyridyl and 3 -pyridyl.
 105. The method ofclaim 101 wherein each of R¹ and R ⁴ is 2 -pyridyl.
 106. The method ofclaim 101 wherein each of R¹ and R ⁴ is 3 -pyridyl.
 107. The method ofclaim 101 wherein at least one of R⁵ and R ⁷ is H.
 108. The method ofclaim 101 wherein: each of R ¹ and R ⁴ is an electron-deficient nitrogenheterocycle; each of R ² and R ³ is H; and each of R ⁵ and R ⁷ is H.109. A method of oligonucleotide ligation, comprising: annealing twoprobes to a target sequence; and forming a phosphodiester bond betweenthe 5′ terminus of one probe and the 3′ terminus of the other probe;wherein one or both probes are labelled with a compound of claim 69 oran energy transfer dye compound of claim 77 .
 110. The method of claim109 wherein the electron-deficient nitrogen heterocycle is selected fromthe group consisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2 -quinolyl,3 -quinolyl, 4 -quinolyl, 2 -imidazole, 4 -imidazole, 3 -pyrazole, 4-pyrazole, pyridazine, pyrimidine, pyrazine, cinnoline, pthalazine,quinazoline, quinoxaline, 3 -( 1,2,4 -N)-triazolyl, 5 -( 1,2,4-N)-triazolyl, 5 -tetrazolyl, 4 -( 1 -O, 3 -N)-oxazole, 5 -( 1 -O, 3-N)-oxazole, 4 -( 1 -S, 3 -N)-thiazole, 5 -( 1 -S, 3 -N)-thiazole, 2-benzoxazole, 2 -benzothiazole, 4 -( 1,2,3 -N)-benzotriazole, andbenzimidazole.
 111. The method of claim 109 wherein theelectron-deficient nitrogen heterocycle is selected from the groupconsisting of 2 -pyridyl, 3 -pyridyl, 4 -pyridyl, 2 -quinolyl, 3-quinolyl, and 4 -quinolyl.
 112. The method of claim 109 wherein each ofR¹ and R ⁴ is an electron-deficient nitrogen heterocycle selected fromthe group consisting of 2 -pyridyl and 3 -pyridyl.
 113. The method ofclaim 109 wherein each of R¹ and R ⁴ is 2 -pyridyl.
 114. The method ofclaim 109 wherein each of R¹ and R ⁴ is 3 -pyridyl.
 115. The method ofclaim 109 wherein at least one of R⁵ and R ⁷ is H.
 116. The method ofclaim 109 wherein: each of R ¹ and R ⁴ is an electron-deficient nitrogenheterocycle; each of R ² and R ³ is H; and each of R ⁵ and R ⁷ is H.